Introduction

Ischemic heart disease (IHD) is a leading cause of death worldwide [1]. MI leads to myocardium loss, pathological remodeling, dysfunctional cardiac function, and heart failure [2]. MI is manifested by myocyte loss, which triggers a cascade of immune-inflammatory pathways and cellular processes such as complement activation, the generation of ROS, and the activation of inflammasomes. The recovery begins with the action of inflammatory cells, which replace the necrotic myocardium with granulation tissue. Also, fibroblasts generate a new collagen matrix, which eventually results in the formation of the post-infarction scar in the infarcted area [3,4,5].

Recent studies have demonstrated that mesenchymal stem cells (MSCs) can reduce inflammation, fibrotic, and injury to the parenchyma, thus contributing to tissue repair [6,7,8,9,10,11,12]. Earlier clinical studies have been shown that transplanted stem cells can promote tissue regeneration via engraftment in the myocardium, not via differentiation into cardiomyocytes [13, 14]. Despite the benefits, MSCs have not translated well into clinical practice because clinical trials have consistently failed to produce conclusive outcomes possibly due to the difficulties in preparing large scale of homogenous cells that can be applicable for therapeutical purposes [15]. In this regard, MSC-like cells derived from pluripotent stem cells (iMSCs) have tremendous advantages owing to their homogeneity and stable growth profile [16, 17]. These properties make it possible to generate a large quantity of clonally derived iMSCs in a scalable manner [18]. Several animal studies using iMSCs have shown significant benefits in tissue regeneration and repair [19,20,69] suggested that HA-iMSC-EVs share pharmacological functions with several drugs associated with cardiovascular diseases/development. HA is an unsulfated glycosaminoglycan with excellent viscoelasticity, high moisture retention capacity, biocompatibility, and hygroscopic properties [70]. Most cells in the body synthesize HA at some point in their cell cycle, implicating it in several fundamental biological processes. Wang et al. reported that HA oligosaccharides improve angiogenesis by upregulating VEGF secretion and myocardial function reconstruction after MI through the polarization of M2-type macrophages [71]. Le et al. also reported that HA-based microrods provide local biochemical and biomechanical signals to reprogram fibroblasts and attenuate cardiac fibrosis [72]. Recent study also reported that Hapln1-expressing epicardial cells were responsible for the processing and organization of HA within the ECM, which has recently been implicated in heart regeneration [73]. Furthermore, HA priming increased the trafficking, adhesion, and internalization of MSC EV into injured target cells, enhancing the therapeutic potency of the EV. HA may act as a bridge between MSC EV and target cells, allowing the EV to be internalized [37]. Together with these previous findings and the results from the present study, HA-iMSC-EVs have potential as a novel cell-free therapeutic option for MI.

Conventionally, most preclinical/clinical investigations on cell therapy for heart diseases have used single-dose delivery, mostly due to the technical difficulties of repeated administration and high lethality [74]. However, EVs lack some of the risks associated with cell-based therapies due to their low immunogenicity, minimal embolism risk, and biocompatibility. Furthermore, EVs can be delivered to the heart via various delivery routes, including intravenous, intracoronary and intramyocardial administration [75]. In the present study, we compared the therapeutic effects of HA-iMSC-EVs administered via various regime (i.e., delivery routes, dosages, and schedules). TTC/Evan’s blue staining showed that the infarct size was significantly reduced by HA-iMSC-EVs compared to that in the vehicle-treated animals, regardless of the delivery route. However, bioluminescence imaging revealed that most of the intravenously injected HA-iMSC-EVs were localized in off-target organs, including the brain, lungs, liver, and kidneys. Therefore, we decided to administer HA-iMSC-EVs via intramyocardial route to reduce the localization and possible side effects in non-cardiac tissues. We also tried to identify whether the repeated injection of HA-iMSC-EVs, considering clinically feasible platforms, could improve cardiac functions in comparison with single injection. However, we found that the repeated injection of HA-iMSC-EVs did not significantly improve systolic and diastolic cardiac functions. In histologic analysis, only capillary densities in the border zone and percent fibrosis of LV wall were significantly improved in the high (20 mg/kg) dose of repeated injection of HA-iMSC-EVs. Considering that the repeated injection of 10 mg/kg of HA-iMSC-EVs didn’t improve capillary densities and percent fibrosis compared with those of single injection of 20 mg/kg of HA-iMSC-EVs, we can infer that (1) the therapeutic concentration of 20 mg/kg in single injection group was set too higher than expected, and (2) the time point of secondary injection (at day 7 after the first one) may not be sufficient to reverse the inflammation and fibrosis, resulting in reduced dose-dependency in some functional parameters. Thus, we are planning to conduct further studies to investigate optimal applications such as HA-iMSC-EVs concentration and dose intervals, in order to enhance the HA-iMSC-EVs treatment regime.

Conclusion

HA-iMSC-EVs improve cardiac repair via multiple cellular mechanisms including promoting capillary growth and attenuating tissue necrosis after MI. This strategy has potential to become an alternative option for cell-free therapeutics for cardiac repair.