PAH 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 PAH is defined as mPAP ≥25 mmHg in children >3 months of age at sea level, mPAP ≥25 mmHg, PCWP <15 mmHg, PVR index >3 WU × m2 . According to the guideline, 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-PAH 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 EPCs. The human expression of VEGFR2 is also known as KDR [16, 19, 24]. CD133 is expressed in early EPCs but absent in mature endothelial cells, which is the surface marker of EPCs [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 EPCs [12, 27, 33]. Therefore, flow cytometry was used in this study to define CD133+/KDR+ cells as EPCs. It has been proved that CD133+/KDR+ cells can differentiate into 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 . Since there is no unified definition and classification of EPCs 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. For patients with EPCs>1.00/μL, the risk of high PAH (>25mmHg) was significantly lower than that for patients with EPCs<1.00/μL (P<0.05), regardless of adjusting gender, age, and BMI or not. However, a significant difference in risk of PAH between EPCs and the middle PAH (20–25mmHg) was not found (P>0.05). PAH severity is negatively correlated with the number of EPCs, suggesting that the reduction of EPCs increases the risk of PAH among patients with CHD. At present, there are no studies on the relationship between EPCs and PAH in children with CHD. In the study by Zhu et al.,[1, 14] the number of EPCs is reduced in idiopathic PAH, which is consistent with our study. Liu et al. also found that EPCs in PAH combined with the chronic obstructive pulmonary disease was decreasing. However, Schiavon et al. concluded that elevated EPCs in patients with end-stage PAH 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 [16, 35]. argue that reduced EPCs levels may lead to endothelial dysfunction in CHD patients, triggering PAH. Due to the continuous damage of pulmonary artery endothelial cells caused by PAH, 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. Sen et al.  believe EPCs to be an important marker of cardiovascular diseases.
Through this study, we believe that in patients with CHD, endothelial cells may be damaged or functioned due to the increase of blood flow or accelerated flow rate in the pulmonary artery 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 PAH and associated with PAP. According to the present study, no difference in the middle PAH 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 PAH in animal studies and achieved good results [40–42]. Lavoie et al.  applied EPCs in the experimental treatment of idiopathic PAH patients and found that the PAP 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 PAH.
According to our study, EPCs may be a protective factor of PAH in children with CHD. Therefore, EPCs may be an effective treatment for patients with CHD complicated with PAH, if the PAP cannot be reduced to a normal level after surgery,.
Strengths and limitation: There are few studies about the relationship between EPCs and PAH in children with CHD. A rigorous experimental design was carried out strictly. Three types of CHD (PDA, ASD, and VSD) are included 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 PAH 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.