2.1 Endothelial progenitor cells (EPCs)
The controversy surrounding EPC definition are beginning from the first observations of blood vessel development in 1917, but after a long period of discussion, the precise definition of EPC are still unclear(Ferkowicz & Yoder, 2005). The most accessed definition of EPC is that progenitor cells are a heterogeneous population which includes different origins and several residing sites at different maturity stages(Yang, Pan, Wang, Qiu, & Mao, 2018). Due to the different features of cells, it is difficult to produce a worldwide accepted characterization of EPCs(Richardson & Yoder, 2011). Additionally, due to the different characterizations of EPCs, some contrary results have emerged(Harper et al., 2019; Toshner et al., 2009; Zhou et al., 2013). Balistreri et al. argued that EPCs are a type of stem cell(Bianconi et al., 2018; Prokopi & Mayr, 2011). In their opinion, progenitor cells are an intermediate stage between multipotent stem cells and mature cells, and both stem cells and progenitor cells have the ability to replicate and differentiate to mature cells, and even have the same biological markers(Brown & McGuire, 2012). EPCs can adhere to matrix molecules such as fibronectin and demonstrate dual positivity to acetylated low-density lipoprotein (acLDL) and Ulex europaeusagglutinin I (UEA-1) lectin(Yang, Pan, Zhao, & Wang, 2013). These types of cells commonly have some common positive protein such as CD34+ and CD133+ (which are two unique markers of hematopoietic stem cells), vascular endothelial growth factor receptor-2 (VEGFR2+, also called kinase insert domain receptor (KDR)/fetal liver kinase-1 (Flk-1), which is an important regulator of development of ECs and vasculature), and von Willebrand factor+ (vWF+), CD31+, CD144+, CD146+, and endothelial nitric oxide synthase+(eNOS+).
EPCs in different classifications are characterized by different abilities and affinities. According to the number of culture days after monocytes are extracted from blood(Hansmann et al., 2011), EPCs can be divided into early-outgrowth EPCs and late-outgrowth EPCs(Pelosi, Castelli, & Testa, 2014; Prokopi & Mayr, 2011). Early-outgrowth EPCs, also called circulatory angiogenic cells (CACs)(Basile & Yoder, 2014; Prater, Case, Ingram, & Yoder, 2007), are a spindle-shaped cell that emerges after culturing for 4-7 days(Murohara, 2010). Its name indicates that this type of EPC possesses little differentiation capacity, and thus, these cells cannot become mature cells(Paneni, Costantino, Kränkel, Cosentino, & Lüscher, 2016). However, early-outgrowth EPCs have a significant capacity to release several cell growth factors and cytokines such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), granulocyte colony stimulating factor (G-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF), and interleukin (IL)-8(Guber, Ebrahimian, Heidari, Eliopoulos, & Lehoux, 2018; J. X. Yang et al., 2018). Due to their unique biological behaviors, early-outgrowth EPCs exert their function mainly by secreting growth factors and cytokines to support the growth of adherent cells and surrounding late-outgrowth EPCs. Except for custom EPC markers, early-outgrowth EPCs also express CD45 and CD14(S. J. Zhang et al., 2006).
Other cell groups emerged after 14-21 days of culture, and these cobblestone-shaped cells are named late-outgrowth EPCs, and also are called endothelial outgrowth cells (EOCs)(S. Liu et al., 2018), although they are different from early-outgrowth EPCs. The late-outgrowth EPCs display a significant capacity to replicate and differentiate to mature ECs. Compared with typical EPCs, late-outgrowth EPCs do not express hematopoietic stem cells markers, but instead, express vascular endothelial-cadherin (VE-cadherin) and CD146(Minami et al., 2015).
Given their biological properties, some researchers recognize that early-outgrowth EPCs and late-outgrowth EPCs are not single types of cells in different stages, but they may differentiate from different cells(Medina et al., 2010). There also exist a small proportion of EPCs that remain in the microcirculation of lung vessels before occurrence of injury(Schniedermann et al., 2010). Therefore, we called this type of EPC resident EPCs (in contrast to circulating EPCs). Resident EPCs display a significant replication and differentiation capacity, suggesting that lung blood and lymphatic ECs may be derived from these cells. However, based on our existing research results, we cannot clearly distinguish this cell type, and therefore, further study is required to discern the nature between resident EPCs and circulating EPCs.