Extracorporeal Cardiac Shock Waves Therapy Improves the Function of Endothelial Progenitor Cells After Hypoxia Injury via Activating PI3K/Akt/eNOS Signal Pathway.

Mingqiang Wang,Zhao Hu,Xingyu Cao,Hongbo Cai,Hongyan Cai,Yiming Ma,Yunke Shi,Tao Guo,Dan Yang

doi:10.3389/fcvm.2021.747497

Abstract

Background: Extracorporeal cardiac shock waves (ECSW) have great potential in the treatment of coronary heart disease. Endothelial progenitor cells (EPCs) are a class of pluripotent progenitor cells derived from bone marrow or peripheral blood, which have the capacity to migrate to ischemic myocardium and differentiate into mature endothelial cells and play an important role in neovascularization and endothelial repair. In this study, we investigated whether ECSW therapy can improve EPCs dysfunction and apoptosis induced by hypoxia and explored the underlying mechanisms.Methods: EPCs were separated from ApoE gene knockout rat bone marrow and identified using flow cytometry and fluorescence staining. EPCs were used to produce in vitro hypoxia-injury models which were then divided into six groups: Control, Hypoxia, Hypoxia + ECSW, Hypoxia + LY294002 + ECSW, Hypoxia + MK-2206 + ECSW, and Hypoxia + L-NAME + ECSW. EPCs from the Control, Hypoxia, and Hypoxia + ECSW groups were used in mRNA sequencing reactions. mRNA and protein expression levels were analyzed using qRT-PCR and western blot analysis, respectively. Proliferation, apoptosis, adhesion, migration, and angiogenesis were measured using CCK-8, flow cytometry, gelatin, transwell, and tube formation, respectively. Nitric oxide (NO) levels were measured using an NO assay kit.Results: Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that differentially expressed genes were enriched in cancer signaling, PI3K-Akt signaling, and Rap1 signaling pathways. We selected differentially expressed genes in the PI3K-Akt signaling pathway and verified them using a series of experiments. The results showed that ECSW therapy (500 shots at 0.09 mJ/mm2) significantly improved proliferation, adhesion, migration, and tube formation abilities of EPCs following hypoxic injury, accompanied by upregulation of p-PI3K, p-Akt, p-eNOS, Bcl-2 protein and NO, PI3K, and Akt mRNA expression, and downregulation of Bax and Caspase3 protein expression. All these effects of ECSW were eliminated using inhibitors specific to PI3K (LY294002), Akt (MK-2206), and eNOS (L-NAME).Conclusion: ECSW exerted a strong repaired effect on EPCs suffering inhibited hypoxia injury by inhibiting cell apoptosis and promoting angiogenesis, mainly through activating the PI3K/Akt/eNOS signaling pathway, which provide new evidence for ECSW therapy in CHD.

Highlights

Coronary heart disease (CHD) is the leading cause of death in adults worldwide [1, 2]
To distinguish the biological pathways that became active in endothelial progenitor cells (EPCs) of the hypoxia and hypoxia +extracorporeal cardiac shock waves (ECSW) groups, we investigated the differentially expressed mRNAs using term enrichment analysis to identify their possible targets using the Kyoto encyclopedia of genes and genomes (KEGG) annotation
We observed that ECSWmediated increase in these proteins in EPCs were reversed by pathway inhibitors, accompanied with an inhibitory effect on EPCs function including migratory, proliferative, adhesive, and tube formation capacities. These results indicated that the inhibition of PI3K/Akt/endothelial nitric oxide synthase (eNOS) signaling pathway in EPCs may be a pathological mechanism for the reduction of endogenous vascular repair in CHD, but ECSW have shown promoting effects on EPCs function after hypoxic injury by activating in PI3K/Akt/eNOS signaling pathway

Summary

Introduction

Coronary heart disease (CHD) is the leading cause of death in adults worldwide [1, 2]. Through tissue cavitation, ECSW produce a series of biochemical effects, including shear stress on cell membranes [9], increased endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) synthesis, and upregulation of vascular endothelial growth factor (VEGF), which attenuates cell apoptosis, inflammatory responses and induces angiogenesis [10,11,12]. Another potential cellular mechanism may involve ECSW inducing endothelial progenitor cells (EPCs) to home to ischemic sites, exerting pro-angiogenesis, anti-oxidative stress, anti-inflammation, and anti-fibrosis effects [13,14,15]. We investigated whether ECSW therapy can improve EPCs dysfunction and apoptosis induced by hypoxia and explored the underlying mechanisms

Methods

Results

Discussion

Conclusion