In this study, miR-28-3p was downregulated in PECs from PE patients and mice, which is opposite to the observation from the plasma of patients with PE [8]. Also, PECs were found to be highly apoptosis-resistant in PE models, which was reversed by upregulation of miR-28-3p through coculturing with miR-28-3p-modified AMSCs and exosomes. Furthermore, APAF1 was the target gene of miR-28-3p, and APAF1/caspase-9 signaling mediated the effects of miR-28-3p on PECs apoptosis. Finally, the effect of exosomal miR-29-3p on PECs was also confirmed in vivo.
For either a single or recurrent episode, PE is thought to lead to pulmonary vascular obliteration that is sufficiently severe to cause pulmonary hypertension [41]. It has been reported that severe pulmonary hypertension results in enhanced proliferative potential of endothelial cells (ECs), fibroblasts, and smooth muscle cells [20]. Also, it is suggested that severe pulmonary arterial hypertension is associated with cancer-like cell futures, such as resistance to antigrowth signals, enhanced proliferation, and evasion of apoptosis [42]. In the current study, the apoptosis level of PECs was downregulated in PECs of both PE patients and mice, which is in agreement with previous studies reporting PE-induced apoptosis-resistance in PECs [43] and pulmonary fibroblasts [44]. Endothelial cells exert vital roles in maintaining vascular integrity and regulating thrombosis, which is essential for vascular homeostasis [45]. The disturbed balance of PECs proliferation and apoptosis in PE may be associated with vascular remodeling since irregular thickening of the vessel is thought of as a main vascular sign of PE [46].
It has been demonstrated that miR-28-3p is a plasma diagnostic marker in PE [8], implying the potential roles of miR-28-3p in PE models. Thus, to further determine the underlying mechanism of PE, the focus was on the effect of miR-28-3p in PECs apoptosis-resistance in PE. In this study, the expression of miR-28-3p was downregulated in PECs of both PE patients and the mouse model along with decreased PECs apoptosis, whereas the upregulation of miR-28-3p by engineered AMSCs and exosomes enhanced the level of apoptosis, indicating the potential roles of miR-28-3p in PECs apoptosis. As a multifunctional regulator, miR-28-3p is involved in various pathological processes. In ovarian cancer, miR-28-3p may play roles in cancer cell motility and invasion [47, 48]. Furthermore, miR-28-3p is found to act as a cellular restriction regulator to suppress replication and infection of human T cell leukemia virus, type 1 [49]. Regarding apoptosis, upregulation of miR-28-3p regulates osteoblasts proliferation and apoptosis to promote fracture healing [50].
Notably, the expression of miR-28-3p detected in PECs disagrees with the report that the plasma level of miR-28-3p elevates in PE patients [8]. The similar paradoxical expression pattern of miRNAs between tissue and plasma has also been reported in prostate cancer [51]. Since the mechanism underlying releasing of miRNAs into the extracellular environment is still not completely clear, this phenomenon may be interpreted by several hypotheses, such as origination from damaged cells [52], microvesicles [39], protective proteins [53], and immunocytes [54]. However, the exact mechanism of this discrepancy should be further explored in the future.
In this study, API5 was identified as a direct target gene of miR-28-3p, and the overexpression of API5 could reverse the effect of the upregulation of miR-28-3p on PECs apoptosis, which indicates API5 signaling plays important roles in miR-28-3p-related PECs apoptosis in PE. Moreover, API5, also known as fibroblast growth factor-2-interacting factor (FIF) or anti-apoptosis clone 11 (AAC-11), was originally found to act as an anti-apoptotic factor in mouse fibroblasts [55], cervical carcinoma cells [56], and liver cells [57]. It is reported that API5 depletion enhances cellular sensitivity to the anti-tumor drug, indicating the anti-apoptotic role of API5 in cancer cell survival [58]. The anti-apoptotic effect of API5 displays an E2 promoter-binding factors (E2F)-dependent pattern [59]. The present study revealed that overexpression of API5 could downregulate apoptosis in exosome-treated PECs, indicating that miR-28-3p/API5 signaling may play important roles in regulating PECs apoptosis in PE.
As one of the important goals in this study, the function of AMSCs-derived exosomes as the vehicle to deliver miR-28-3p to PECs was investigated. The present study suggested that miR-28-3p was highly expressed in modified AMSCs and exosomes, and miR-28-3p expression was also upregulated in PECs after coculturing with exosomes, thereby alleviating apoptosis-resistance of PECs. Together, these results suggest that AMSC exosomes are operable and an efficient way to transport miRNA to PECs. As a promising approach, the specificity, accessibility, and stability of exosome-carried miRNAs make them a high-efficiency way to regulate cellular functions and activities [37], which is widely applied in multiple studies. For example, AMSC-originated exosomal miR-122 can enhance hepatocellular carcinoma chemosensitivity [60]. In addition, AMSC-derived exosomes elevate miR-17 expression in hepatic macrophages, thereby ameliorating acute liver failure in mice [61]. Therefore, AMSC exosome-based transportation of miRNAs may be a promising therapeutic strategy.
In conclusion, in this study, miR-28-3p was downregulated in PECs from both PE patients and mice, which is opposite to the observation from the plasma of patients with PE [8]. Also, PECs were found to be highly apoptosis-resistant in PE models, which could be reversed by the miR-28-3p upregulation through coculturing with miR-28-3p-modified AMSCs or exosomes. Furthermore, API5 was the target gene of miR-28-3p, and API5 mediated the effects of miR-28-3p on PECs apoptosis. Finally, the effect of exosomal miR-29-3p on PECs was also confirmed in the PE mice model in vivo. The results suggested that miR-28-3p could ameliorate PE-associated apoptosis-resistance in PECs through targeting API5, and AMSCs-derived exosomes are an ideal way to deliver functional miRNA to PECs for PE treatment. Thus, this study may provide a novel strategy to treat PE disease.