Research article
Extracellular vesicles derived from macrophage promote angiogenesis In vitro and accelerate new vasculature formation In vivo

https://doi.org/10.1016/j.yexcr.2020.112146Get rights and content

Highlights

  • Ischemia is the partial or complete blockage of blood supply to tissues.

  • VEGF, Wnt3a and miR-130a were more abundant in the MAC-EVs than cells.

  • MAC-EVs treatment resulted in increased endothelial cellular proliferation, migration, and tube formation in vitro.

  • In vivo assay results revealed that MAC-EVs increased the formation of new and larger blood vessels.

  • MAC-EVs have the potential to induce angiogenesis and could serve as alternative angio-EVs for ischemic diseases.

Abstract

Background

Ischemia is the partial or complete blockage of blood supply to tissues. Extracellular vesicles (EVs) are emerging as a therapeutic tool for ischemic diseases. Most EV-based ischemia therapies are based on various stem cells. Here, we propose an alternative cell source for the isolation of pro-angiogenic EVs.

Methods

EVs were isolated from a mouse macrophage cell line (Raw 264.7). The characteristic features of the macrophage-derived EVs (MAC-EVs) were assessed using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting (WB) analysis. WB and qRT-PCR were performed to identify the pro-angiogenic VEGF and Wnt3a proteins and microRNAs (miR-210, miR-126, and miR-130a) in the MAC-EVs. In vitro and in vivo Matrigel plug assays were performed to investigate the capacity of the MAC-EVs for tube (blood vessel-like) formation and new blood vessel formation and assessed by histology.

Results

The MAC-EVs was positive for ALIX and negative for calnexin, with a round shape and an average size of 189 ± 65.1 nm. WB and qRT-PCR results revealed that VEGF, Wnt3a and miR-130a were more abundant in the MAC-EVs than cells. MAC-EVs treatment resulted in increased endothelial cellular proliferation, migration, and tube formation in vitro. In vivo assay results revealed that MAC-EVs increased the formation of new and larger blood vessels in the Matrigel plug of mice compared to the formation in the control group.

Conclusion

Our results suggest that MAC-EVs have the potential to induce angiogenesis in vitro and in vivo, could serve as a pro-angiogenic alternative for ischemic diseases.

Introduction

Angiogenesis is the development of blood vessels from pre-existing vasculature. It is mediated by a complex multistep process comprising a series of cellular events such as endothelial cell (EC) proliferation, migration, or invasion into tissues and new capillary sprouting and maturation [1,2]. Angiogenesis plays critical roles in human physiology ranging from reproduction and fetal-development to tissue repair and wound healing [3,4]. Ischemia occurs when blood flow to tissues or organs is reduced, preventing the supply of sufficient oxygen and can occur anywhere in the body such as myocardial ischemia, limb ischemia, and cerebrovascular ischemia. The reduced blood flow to tissues or organs result in partial or complete damage [[5], [6], [7]]. Myocardial ischemia is responsible for over 15% of mortality each year [8] and limb ischemia has a 30% amputation rate and a 25% mortality rate [9]. Despite advancements in surgical, endovascular, and anesthesia-based techniques, treatment options for patients with ischemia are limited due to the current understanding of progenitor cell biology and cell-based therapies [9,10].

In the last two decades, stem cell-based therapy has emerged as an option for the treatment of ischemic diseases. Several stem cell types including embryonic stem cells, neural stem/precursor cells, mesenchymal stem cells from several tissue sources, and induced pluripotent stem cells have been applied in preclinical and clinical research [9,11,12]. Recently, it was hypothesized that the therapeutic effects of cells occur primarily via secreted factors including extracellular vesicles (EVs) [13,14]. EVs, which include exosomes and microvesicles, are membrane-bound nano-vesicles released by almost all cells [[15], [16], [17]]. EVs are found in most biological fluids such as urine, plasma, cerebrospinal fluid, saliva, and breast milk as well as in cell culture conditioned media [[18], [19], [20], [21]]. They contain lipids, proteins, microRNAs (miRNAs), mRNAs, noncoding RNAs (ncRNAs), and DNAs [16,22]. Biological information can be transported to nearby cells or distant cells via EVs, a feature that has generated significant interest in scientific communities [23,24].

Currently, the research on EVs released from stem cells has been extensively focused on mesenchymal stem cells (MSCs). MSC-EVs have been explored as a treatment option for various ischemic diseases including hindlimb ischemia [13,25,26], myocardial ischemia [27,28], and cerebral ischemia [29,30] by promoting angiogenesis through various proteins and miRNAs. MSC-EVs have shown potential as EV-based therapeutics for ischemic diseases [14]. However, the isolation of MSC-EVs involves several challenges such as cell expansion, which takes several days or weeks, the passage number of the MSCs directly corresponding to reduced cell viability [31], heterogeneity within the MSCs, a variable capacity for neovascularization [32], and the invasiveness of isolating MSCs from bone marrow. All these factors are directly involved in the isolation of EVs from MSCs. Therefore, identifying potential alternative cell-derived pro-angiogenic EVs is urgently required.

Here, we propose macrophage cells as an alternative to MSC-derived EVs. Because the isolation of macrophage cells is easier than that of stem cells, macrophages can be isolated from blood less invasively. Several studies have described the role of macrophages in angiogenesis and regeneration [[33], [34], [35]]. However, the pro-angiogenic effects of macrophage-derived EVs (MAC-EVs) are unknown. In this study, we, for the first time, investigated the effect of EVs isolated from the supernatant of cultured macrophages using endothelial in invitro and in vivo mouse models to find a potential alternative EV to stem cell derived EVs.

Section snippets

Cell culture

The murine macrophage cell line Raw 264.7 and murine endothelia cell line SVEC4-10EHR1 were purchased from American Type Culture Collection (Manassas, Virginia, USA). The cells were cultured in DMEM-high glucose (HyClone, Logan, UT, USA), supplemented with 10% EV-depleted fetal bovine serum (FBS) (HyClone) (18 h, at 120,000×g and 4 °C) and 1% penicillin-streptomycin (Gibco, Carlsbad, CA, USA), at 37 °C with 5% CO2.

Isolation of extracellular vesicles from macrophage cells

Extracellular vesicles were isolated from the culture medium of macrophage cells

Isolation and characterization of MAC-EVs

MAC-EVs were isolated from the macrophage culture supernatant as described above. First, we confirmed the presence of the EV protein marker ALIX (a cytoplasmic protein defined as a regulator of the endo-lysosomal system). EV biomarker was found to be abundant in the MAC-EVs compared to their parent cells. In addition, a cell-positive and EV-negative biomarker calnexin was tested. Our results showed that calnexin was present only in the cell lysate and absent in the MAC-EVs (Fig. 1A). We also

Discussion

In the present study, MAC-EVs were shown to have pro-angiogenic capabilities, possibly due to their cargo such as proteins or miRNAs working individually or synergistically by inducing proliferation, migration, and tube formation in ECs. Additionally, MAC-EV treatment accelerated neovascularization in mouse by increasing the number of blood vessels and inducing the formation of larger blood vessels. The enrichment of EV marker protein such as ALIX was confirmed and the absence of the cell

Conclusion

We successfully isolated EVs from macrophages and identified the pro-angiogenic cargos (VEGF, Wnt3a, miR-210, miR-126, and miR-130a). Furthermore, our data demonstrated that MAC-EVs can increase EC proliferation, migration, and tube formation in vitro. We also showed that MAC-EVs with Matrigel could enhance the retention times at the injection site in mice. Further testing showed that MAC-EVs can increase neovascularization of Matrigel in vivo by inducing the formation of new blood vessels and

CRediT authorship contribution statement

Prakash Gangadaran: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Writing - original draft. Ramya Lakshmi Rajendran: Conceptualization, Data curation, Formal analysis, Investigation, Writing - original draft. Ji Min Oh: Investigation, Methodology, Writing - review & editing. Chae Moon Hong: Methodology, Writing - review & editing. Shin Young Jeong: Resources, Writing - review & editing. Sang-Woo Lee: Resources, Writing - review & editing. Jaetae Lee:

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Republic of Korea (Grant number: NRF-2019R1I1A1A01061296). This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant number: HI15C0001).

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    These authors contributed equally to this study.

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