Extracorporeal cardiac shock wave stimulation enhances the therapeutic efficacy of intravenously delivered endothelial colony-forming cells via PI3K/AKT signaling in a rat myocardial infarction model

Abstract

Background: Extracorporeal cardiac shock wave (ECSW) therapy enhances the function of endothelial colony-forming cells (ECFCs), but whether it can serve as a preconditioning strategy to enhance myocardial infarction (MI) therapy remains unclear. This study investigated the efficacy and mechanism of intravenously delivered ECSW-preconditioned ECFCs (SW-ECFCs) in a rat MI model. Methods: ECFCs were isolated from the bone marrow of ApoE-/- rats and fully characterized. RNA sequencing of control ECFCs versus SW-ECFCs revealed significant enrichment of the PI3K/AKT pathway. We therefore performed a series of in vitro functional assays on these cells, including Transwell migration, Matrigel tube formation, CCK-8 proliferation, flow cytometric apoptosis analysis, and VEGF-A ELISA. The role of the PI3K/AKT pathway was interrogated using the inhibitor LY294002. Subsequently, an acute MI model was established in ApoE-/- rats via left anterior descending coronary artery ligation. Rats were randomized into four groups: MI+PBS, MI+ECFCs, MI+SW-ECFCs, and MI+LY294002-pretreated SW-ECFCs (LY-SW-ECFCs), with sham-operated rats as controls. Comprehensive evaluations included echocardiography, serum injury biomarkers, TTC, and histopathological (H&E, Masson) staining, immunohistochemical detection of cardiomyocyte apoptosis and p-eNOS, immunofluorescence assessment of ECFC homing and vascular markers (CD31, α-SMA, VEGF-A), tissue/plasma nitric oxide measurement, and Western blot analysis of PI3K/AKT signaling proteins. Results: Transcriptomic analysis revealed significant enrichment of the PI3K/AKT pathway in SW-ECFCs. Functionally, ECSW enhanced ECFCs migration, tube formation, proliferation, and VEGF-A secretion, while reducing apoptosis; these effects were largely abolished by PI3K inhibition. In vivo, serum levels of CK, CK-MB, and LDH were significantly elevated in all MI groups compared to the Sham group (P<0.01), indicating comparable initial injury. However, no significant differences were observed among treatment groups (P>0.05). SW-ECFCs transplantation significantly improved cardiac function, reduced infarct size, fibrosis, and apoptosis, and enhanced angiogenesis (P<0.05). These benefits were associated with increased levels of p-AKT, p-eNOS, and BCL-2 protein as well as nitric oxide content, while suppressing the expression of cleaved caspase-3 (P<0.05). Crucially, all these therapeutic benefits were largely abolished by PI3K inhibition. Conclusion: In conclusion, this study demonstrates that preconditioning ECFCs with ECSW significantly enhances their therapeutic efficacy for myocardial infarction, improving both cardiac function and structural repair. These benefits are mediated primarily through activation of the PI3K/AKT signaling pathway, which augments cell homing, paracrine activity, and survival, thereby providing a novel and promising strategy for cardiac regeneration.

1. Introduction

Acute myocardial infarction (AMI), characterized by acute persistent ischemia of major coronary arteries, leading to cardiomyocyte necrosis, remains the leading global cause of mortality [1]. Hypoxia-induced endothelial cell injury represents a fundamental pathogenic mechanism in coronary artery disease progression. Contemporary revascularization strategies, such as percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG), significantly improve symptoms and prognosis for many patients by enhancing myocardial perfusion. However, these methods are often insufficient to prevent recurrent myocardial ischemia and significantly improve the quality of life in patients with refractory angina [2, 3]. This unmet clinical need highlights the necessity for innovative treatments, such as stem cell therapies, to stimulate angiogenesis and thereby improve coronary blood flow and prevent further tissue damage.

Stem cell-based regenerative medicine has shown considerable potential for cardiac repair, as evidenced by clinical trials and research that demonstrate its effectiveness in treating heart diseases. Endothelial colony-forming cells (ECFCs), a well-defined population of endothelial progenitor cells derived from bone marrow or peripheral blood, exhibit therapeutic promise owing to their capacity to migrate to ischemic myocardium. At the injury site, ECFCs contribute to vascular repair through direct incorporation into nascent endothelium and/or paracrine-mediated angiogenesis [4–6], highlighting their essential role in post-infarction microvascular reconstitution. However, patients with coronary artery disease exhibit not only a reduced number of ECFCs and impaired migratory capacity [7] but also limited intrinsic myocardial repair ability [8, 9]. Consequently, insufficient recruitment of endogenous ECFCs to the infarcted area hinders endothelial repair, limiting their therapeutic utility. This inherent impairment in vascular repair underscores the necessity of employing exogenous strategies to promote vascular regeneration in ischemic cardiomyopathy. Although targeted delivery of ECFCs (e.g., via infarction border zone transplantation) has shown promising results in treating myocardial infarction, its application is constrained by several challenges [10–12]. Despite encouraging preclinical outcomes, the low engraftment efficiency and poor survival of transplanted ECFCs remain major obstacles to clinical translation [12, 13]. Current preconditioning methodologies, such as hypoxia exposure, genetic modification, and hydrogel encapsulation, often have limited efficacy and involve procedurally complex protocols [14]. Therefore, to advance therapy, it is necessary to develop clinically applicable and safe preconditioning strategies that can robustly enhance the viability, functionality, and regenerative potential of ECFCs.