3.12 Preclinical research on EPCs in PAH
Because EPCs have numerous capacities such as proliferation, migration, and adhesion to protect the normal structure of ECs and PASMCs, scientists use EPCs to attenuate and reverse PAH. Zhao et al. injected fluorescently labeled EPCs into monocrotaline (MCT)-induced PAH mice, the EPCs integrated into the distal pulmonary artery endothelium and limited the progression of PAH through differentiation to mature ECs and alleviation of neointima formation(Yang et al., 2013). Zhao et al. also detected EPC-derived proangiogenic growth factors (such as IL-8 and adrenomedullin) in the adjacent area of EPCs through autocrine and paracrine methodology. Zheng et al. found that EVs of EPCs transport mRNA to ECs, and express a series of proteins such as chemokines, pathway proteins, and growth factors to enhance EC proliferation and migration and decrease angiogenesis(Deregibus et al., 2007; Ingram et al., 2004; X. Li et al., 2016). Zhao et al. used EPCs to treat monocrotaline (MCT)-induced PAH mice at two different time points to explore the effect of EPCs on different disease stages. The results seemed inspiring, as compared with the MCT group, the hemodynamic improvement in both groups was significant, but both treatment groups still had a higher mean pulmonary arterial pressure (mPAP) than the control group, indicating that EPCs can prevent but cannot reverse the occurrence of PAH. Zhao et al. also investigated the microstructure of the pulmonary artery under EPC therapy. They stained the pulmonary artery with fluorescent microspheres and alpha-smooth muscle actin (α-sma) to visualize the structure of PASMCs and microvasculature perfusion. The results showed that the 21-day and 35-day groups dramatically increased their microvascular perfusion and microvasculature after EPC treatment(Y. D. Zhao et al., 2005).
As a nonoral therapy, use frequency is also an important indicator of feasibility. Therefore, Harper et al. and Zhou et al. investigated the stay time and stay location after EPC transplantation. Harper et al. selected cells expressing CD34, CD306, CD146, and CD45 by fluorescence-activated cell sorting (FACS) and transfected adenoviral vectors carrying the luciferase and GFP reporter gene. The immunofluorescence results showed that at 1 h and 7 h after EPC transplantation, most of the luminescence was located in the chest region, and there was little or no luminescence in other areas, the luciferase results were same. Compared with the control group, the concentration of EPCs in the lung was two times higher than that in the spleen. This shows that EPCs tend to aggregate in damaged endothelial tissues, but rarely aggregate in integrated tissues. According to the above evidence, EPC therapy seems to be a targeting therapy that has little impact on other organs, at least in concentration relationships(Harper et al., 2019; Zhou et al., 2013). However, the experiment by Zhou et al. produced some opposite results. He found that the retention rate of EPCs was still nearly 50% 25 days after transplantation(Zhou et al., 2013). These contrary results were caused by different screening conditions during FACS.
In order to improve the effect of EPC therapy, scientists use gene editing technology. Wei et al. used Ad.CMV-human endothelialNO synthase enzymes (Ad.CMV-heNOS), Ad.CMV, or Ad.CMV-enhanced green fluorescent protein (Ad.CMV-EGFP) to infect EPCs for 72 h. Two weeks after the injection, the systolic arterial pressure (ASP) of mice did not significantly change, but the systolic pulmonary artery pressure (sPAP) significantly decreased in both the simple EPC group and the heNOS-EPC group. Furthermore, the heNOS-EPC group was significantly lower than the vector-EPC-treated group and the normal EPC-treated group. The histology results also showed the same trend, with a significant reduction in the number of muscular pulmonary arteries and thickness of the muscular coat(Wei et al., 2013).
Cao et al. selected the human hypoxia inducible factor-1 alpha (hHIF-1α) gene and transfected it into EPCs. Compared with the EPC group and blank group, hHIF-1-EPCs significantly reversed the vascular remodeling, and decreased the sPAP, mPAP, and right ventricular/left ventricular + septum (RV/LV+S). Xu et al. chose to disturb the metabolic process of the pulmonary artery to control the progression of PAH. They used lentiviral vectors to inhibit the expression of E2F transcription factor 1 (E2F1) and subsequently increase oxidative metabolism, endothelial differentiation, vascular repair, and decrease the pyruvate dehydrogenase kinase 4/2 (PDK4/2) expression. The metabolic level was measured by the oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and lactate level. The results showed that E2F1-/- mice had a greater OCR, a lower ECAR, and a lower level of lactate (which represents a lower anaerobic respiration level), indicating that E2F1-/-EPC enhanced vascular growth, reduced infarct size, and improved vascular function through increasing the oxygen utilization rate of pulmonary ECs(Cao et al., 2015; S. Xu et al., 2018).
Dysfunctions of the prostacyclin (PGI) pathway are also an important factor in the occurrence of PAH. Therefore, Zhou et al. tried to engineer the cyclooxygenase-prostacyclin (COX-PGI) pathway to improve the effect of EPCs. The engineered EPCs significantly attenuated the RVSP increase, RV hypertrophy, and intimal and medial smooth muscle layer cell proliferation, and enhanced adventitial pulmonary vessel wall apoptosis(Zhou et al., 2013).