Large extracellular vesicles from induced pluripotent stem cell-marrow stem cells enhance limb angiogenesis via ERK/MAPK

Abstract

Aim: This study aims to investigate the effects of large extracellular vesicles (EVs) induced by pluripotent stem cell-derived mesenchymal stem cells on lower limb ischemic disease and explore its potential mechanisms. Materials & methods: The pathology of muscles was accessed by H&E staining and immunofluorescence staining. In vitro, we conducted wound-healing assay, tube formation assay, RT qPCR, ELISA, RNA sequencing and proteomic analysis. Results: iMSCs-lEVs alleviated the injury of ischemic lower limb and promoted the recovery of lower limb function. In vitro, iMSCs-lEVs promoted the proliferation, migration, and angiogenesis of HMEC-1 cells by regulating the ERK/MAPK signing pathway. Conclusion: This study demonstrated that iMSCs-lEVs promoted endothelial cell angiogenesis via the ERK/MAPK signaling pathway, thereby improving function after lower limb ischemic injury.

Plain language summary

Article highlights.

• The therapeutic potential of large extracellular vesicles derived from induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs-lEVs) in promoting recovery after lower limb ischemia has rarely been investigated so far.

• Improving the functional status of endothelial cells may be effectively alleviate the symptoms of lower limb ischemia.

• iMSCs-lEVs could restore blood flow perfusion in ischemic tissues of mouse lower limbs and improved their function following ischemia.

• iMSCs-lEVs promoted endothelial cell proliferation, migration and tube formation by regulating the ERK/MAPK signaling pathway.

• These findings reveal the molecular mechanisms underlying the application of iMSCs-lEVs on mast cells and provide a novel therapeutic strategy for pain caused by tendinopathy.

1. Background

Peripheral vascular disease (PVD) is a prevalent clinical condition that can cause critical limb ischemia as its most severe manifestation [1–3]. Despite interventions, patients with critical limb ischemia often face a poor prognosis, compounded by the presence of comorbidities that render them unsuitable for revascularization treatment [4,5]. Given the limited treatment options, there is a growing interest in developing novel strategies that aim to improve tissue perfusion by stimulating angiogenesis and bypassing occluded vessels. Such approaches have received considerable attention in the scientific community, as evidenced by the growing number of studies investigating the underlying mechanisms of angiogenesis for the treatment of limb ischemia [6].

In recent years, with the advancement of stem cell research and clinical applications, bone marrow stem cell (BMSC) transplantation into the muscles of patients with limb ischemia has achieved significant results in promoting angiogenesis and restoring blood flow in the affected limbs [7,8]. However, the use of BMSCs in clinical settings is limited by their potential tumorigenicity and immunogenicity. Therefore, researchers have focused on the therapeutic potential of extracellular vesicles (EVs), which are important paracrine components of BMSCs [9]. Several studies have reported the ability of BMSC-derived EVs to stimulate angiogenesis and promote tissue repair, highlighting their potential as an alternative to cell-based therapies [10,11].

Extracellular vesicles (EVs) are a type of secretion produced by most cells, which contain various types of nucleic acids, lipids and proteins from the parent cells [12,13]. In accordance with the MISEV 2018 guidelines, EVs can be divided into small EVs (sEVs, <200 nm) and large EVs (lEVs, >200 nm) based on their particle size [14]. Our previous research has shown that sEVs derived from induced pluripotent stem cells (iPSCs) can effectively promote angiogenesis in ischemic tissues and reduce tissue damage after ischemic injury [15]. Recently, many studies have suggested that lEVs are capable of exerting similar biological functions as sEVs. For instance, Cosenza S, et al. reported that lEVs derived from MSCs activate T and B lymphocytes to exert anti-inflammatory effects [10]. Shahin HI et al. identified that lEVs released by amniotic epithelial cells under oxidative stress initiate inflammation in uterine cells related to parturition [16]. However, the efficacy of lEVs in the treatment of limb ischemia and the mechanisms behind it remain unclear.

In this study, we investigate the therapeutic potential of lEVs derived from induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs-lEVs) in the model of hindlimb ischemia. Initially, we isolated small EVs (sEVs) and large EVs (lEVs) from iPSCs and compared their therapeutic efficacy with sEVs as a reference. Our results demonstrated that lEVs significantly enhanced blood vessel regeneration in the mouse model of hindlimb ischemia. Subsequent in vitro experiments, such as RT-qPCR, ELISA and western blot, revealed that lEVs induced the expression of angiogenesis-related genes and promoted protein secretion. Furthermore, our data indicated that the therapeutic effects of iPSC-derived EVs were mediated through the ERK/MAPK signaling pathway. Collectively, our study provides novel insights into the potential of iPSC-derived lEVs as a promising therapeutic strategy for promoting angiogenesis in ischemic hindlimbs.

2. Materials & methods

2.1. Derivation & culture of iPSC-derived MSCs

The generation of hiPSC-MSCs (iMSCs) were according to our previous study [17]. The serum-free ncMission hMSC Medium (RP02010, Nuwacell Biotechnologies, China) was used to cultivate iMSCs, and every 2 days, the culture medium was collected and replenished. After the cell confluency reached roughly 80%, iMSCs were passaged. The following tests were conducted using iMSCs from passages 5 through 10.

2.2. Isolation of iMSCs-derived large EVs (lEVs) & small EVs (sEVs)

By using ultracentrifugation in conjunction with serial centrifugation, iMSCs-derived lEVs and sEVs were separated using previously published techniques [18]. In summary, dead cells were extracted from the iMSCs culture media by centrifuging it for 10 min at 300×g (4°C). The supernatant was then centrifuged for 20 min at 2000 ×g (4°C). The lEVs were then pelleted by centrifuging the supernatant for 30 min at 10,000 ×g (4°C). Next, the supernatant from which lEVs have been isolated was then passed over a 0.22 μm disposable membrane (Millipore, MA, USA) for filtering, and the sEVs were pelleted by ultracentrifuging for 70 min at 100,000 ×g (4°C).

2.3. Transmission electron microscopy

A 10 μl of lEV or sEV suspensions were placed onto a 400 mesh formvar carbon-coated grid for transmission electron microscopy (TEM) examination, and the grid was allowed to dry for 20 min. The grids were then cleaned with PBS, fixed for five min in 1% glutaraldehyde, rinsed again with deionized water, then stained for five min with uranyl oxalate. The microstructure of lEVs or sEVs was photographed using a TEM (Hitachi H-7650, Tokyo, Japan) after they had dried.

2.4. Size distribution & particle concentration

Using the ZetaView device (Particle Metrix, Meerbusch, Germany), nanoparticle tracking analysis (NTA) was used to determine the size distribution and particle concentration of sEVs and lEVs. Samples were given under a regulated flow after being diluted in PBS at the appropriate ratios. ZetaView Software 8.03.04.01 (Particle Metrix) provided the size distribution and particle concentration data.

2.5. Protein concentration assay

The protein concentrations of lEVs and sEVs were determined using the Pierce BCA Protein Assay Kit (Cat#23225, Thermo Fisher Scientific, MA, USA). Briefly, lEVs and sEVs were lysed with RIPA lysis buffer (Beyotime, Jiangsu, China), and proteins were extracted manually. Next, load 10 μl protein sample into each well of a 96-well plate and add 200 μl of working reagent. The plate was incubated at 37°C for 30 min.

2.6. Culture of HMEC-1 cells

Human microvascular endothelial cells (HMEC-1 cells) were purchased from American Type Culture Collection (ATCC) and cultured in MCDB 131 medium (Gibco, UT, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), 10 ng/ml epidermal growth factor 10 mM L-glutamine and 1 μg/ml hydrocortisone.

2.7. Animal model & experimental design

All animal experimental procedures were approved by the Animal Research Committee of Shanghai Jiao Tong University Affiliated Sixth People's Hospital (SYXK2021-0028, Shanghai, China). Based on the previously established modeling methods, a mouse model of hindlimb ischemia was created [19]. Male C57/BL6 mice at 8 weeks of age, weighing approximately 20 g-25 g, were divided into four groups: sham-operated group, PBS intervention group, iPSC-MSC-sEVs (1 × 10¹⁰ particles/ml) intervention group and iPSC-MSC-lEVs (1 × 10⁹ particles/ml) intervention group, with five mice in each group. Immediately after the surgery, a multi-point injection method (4 injection sites) was used to inject 100 ul of PBS, iPSC-MSC-lEVs and iPSC-MSC-sEVs into the ischemic left quadriceps muscle of the mice. Laser Doppler perfusion imaging was used to detect the degree of blood flow recovery in the ischemic hindlimb at 1, 7, 14 and 21 days postoperatively. Dynamic injury scoring were conducted to evaluate the recovery effect of iMSC-lEVs on ischemic limbs in mice. Mice were euthanized by excessive anesthesia, and the quadriceps femoris of mice in each group were collected for H&E staining and immunohistochemical staining to observe the morphology of the ischemic quadriceps femoris, as well as microvascular formation.

2.8. Histological evaluation & immunohistochemistry

At days 7, 14 and 21, the mice were sacrificed via intraperitoneal injection of an overdose of chloral hydrate. The quadriceps muscle was separated, preserved with 4% paraformaldehyde overnight at 4°C, and then embedded in OCT. To reduce autofluorescence, tissue sections were pre-incubated with sodium borohydride (1 mg/ml in PBS), then incubated overnight at 4°C with antibodies against CD31 (1:100; Abcam) and CD34 (1:100; Abcam), followed by an hour of incubation with secondary antibodies (1:200; Abcam). The nuclei were stained with DAPI (2 μM, Sigma).

2.9. Endothelial cell proliferation assay

The Cell Counting Kit-8 (CCK-8) assay (Dojindo, Kyushu Island, Japan) was used to measure cell proliferation. HMEC-1 cells were seeded at a density of 5 × 10⁴ cells/ml (100 μl/well) on a 96-well plate. After 12 h of quiescence, cells were treated with iMSCs-sEVs (1 × 10⁹ particles/ml) and iMSCs-lEVs (1 × 10⁸ particles/ml) or control medium. At days 0, 1, 2, 3 and 4, 10 μl of CCK-8 solution was added to the medium and incubated at 37°C for 2 h.

2.10. Endothelial cell migration assay

The scratched wound test was performed to determine the migratory effect of iMSCs-sEVs and iMSCs-lEVs on HMEC-1 cells. To achieve a confluent monolayer, 2 × 10⁵ cells were seeded onto 12-well plates and maintained at 37°C. Next, these confluent monolayers were ‘scratch’-wounded with a p200 pipet tip. The media was withdrawn and cleaned with PBS to remove debris and smooth the edge of the scratch, then replaced with iMSCs-sEVs (1 × 10¹⁰ particles/ml), iMSCs-lEVs (1 × 10⁹ particles/ml), or control medium. The level of wound closure was assessed as follows: Migration Index = the migrated cell surface area/total surface area.

2.11. Tube formation assay

Tube formation in vitro was assessed using growth factor-reduced Matrigel (BD Biosciences). At least 30 min before the experiment, 96-well plates were covered with Matrigel. Next, 2 × 10⁴ HMEC-1 cells were seeded on the plated Matrigel with control medium, iMSCs-sEVs (1 × 10¹⁰ particles/ml) and iMSCs-lEVs (1 × 10⁹ particles/ml). The capillary-like structures were observed by microscope.

2.12. Transwell assay

The Transwell test was performed to assess the effect of iMSCs-sEVs and iMSCs-lEVs on HMEC-1 cell invasion. To begin, 30 μl of Matrigel (Corning) was mixed with PBS and placed in the upper chamber of a 24-well transwell plate. Following that, the transwell plates were incubated in the incubator for 4 h. The upper chamber was seeded with 4 × 10⁴ cells HMEC-1 cells with iMSCs-sEVs (1 × 10¹⁰ particles/ml) and iMSCs-lEVs (1 × 10⁹ particles/ml) per well. 24 h later, the cells that had moved to the bottom of the chamber were fixed and stained with 1% crystal violet solution before being examined under a microscope.

2.13. Blood flow measurements & the functional scores

Laser-Doppler Perfusion Imaging (Moor Instruments, Devon, UK) was used to assess blood flow recovery after femoral artery excision on days 0, 7, 14 and 21 post-injection. The digital color-coded images were examined by comparing the blood flow ratios of the ischemic (left) and undamaged (right) legs, expressed as percentage perfusion. The severity of the ischemia was determined by an assessment of ambulatory impairment (3 = dragging of foot, 2 = no dragging but no plantar flexion, 1 = plantar flexion and 0 = flexing the toes to resist gentle traction on the tail) and tissue damage (4 = any amputation, 3 = severe discoloration or subcutaneous tissue loss or necrosis, 2 = moderate discoloration, 1 = mild discoloration and 0 = no difference from the right hindlimb), as previously described [20]. Prior to anesthesia, all mice's limb function and blood flow were measured simultaneously.

2.14. Flow cytometry analysis

The iMSCs were treated with 1% bovine serum albumin (BSA, Gibco) to inhibit non-specific antigens. Then, iMSCs were treated with the following antibodies at 4°C for 30 min. CD29-PE (1:100; 561795, BD Biosciences), CD34-APC (1: 100; 560940, BD Biosciences), CD44-FITC (1: 100; 560977, BD Biosciences), CD73-PE (1: 100; 561014, BD Biosciences), CD90-PE (1:100; 328109, Biolegend), CD105-FITC (1:100; 561443, BD Bio sciences), CD133-PE (1: 100; 130-080-801, Miltenyi Biotec), CD146-PE (1:100; 561013, BD Biosciences), CD45-FITC (1:100; 560976, BD Bio sciences) and HLADR-PE (1:100; 560943, BD Biosciences). After that, two 1% BSA washes were performed on the uncombined antibodies. The CytoFLEX flow cytometer (Beckman Coulter Life Sciences, USA) was used to assess the cells. Data were processed by FlowJo Software (TreeStar, OR, USA).

2.15. Uptake of iMSCs-sEVs / lEVs in vitro & in vivo

In the HMEC-1 cells in vitro experiment, sEVs or lEVs were stained with DiI fluorochrome (Thermo Fisher, USA) and incubated and washed as previously described. Then, DiI-labeled sEVs/lEVs were put into culture media and cultured with HMEC-1 cells for 6 h at 37°C. After discarding the culture medium, the cell cytoskeleton was stained with FITC-labeled phalloidin (C1033, Beyotime) and the cell nuclei were stained with DAPI (2 μM, Sigma) before capturing images using a fluorescence microscope (Leica, DM6B, Germany). In vivo experiments, sEVs or lEVs were incubated with DiR for 15 min at room temperature, then ultracentrifuged at 100,000 g in PBS. Rats' left quadriceps were injected with 100 μl DiR-labeled sEVs or lEVs. The DiR fluorescent signals were detected by an IVIS Spectrum imaging system (PerkinElmer, USA) 12 h after injection.

2.16. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis

Total RNA was extracted using QIAzol Lysis Reagent and RNeasy Mini Columns (Qiagen, CA, USA). The concentration and purity of total RNA were determined using a NanoDrop Spectrophotometer (ThermoFisher Scientific). The RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, CA; Cat#K1622) was used to complete the reverse transcription. RT-qPCR was then carried out utilizing the ABI Prism 7900HT Real Time System (Applied Biosystems, CA, USA) and the SYBR green kit (Roche Applied Science; Cat# 04913850001). The primer sequences used in this work are presented in Supplementary Table S1.

2.17. Enzyme-linked immunosorbent assay (ELISA)

To measure angiogenic trophic factors released by HMEC-1 cells, 8 × 10⁵ cells were seeded onto six-well plates and grown with iMSCs-sEVs (1 × 10¹⁰ particles/ml), iMSCs-lEVs (1 × 10⁹ particles/ml), or control media for 36 h. ELISA kits (all from Westang Bio-tech, Shanghai, China) were used to detect the concentrations of hVEGFA, hEGF and hbFGF.

2.18. Western blot analysis

The protein samples from lEVs and sEVs were collected with RIPA lysis buffer (Beyotime, Jiangsu, China). The western blot analysis was carried out as previously described [21]. Protein extracts were resolved using SDS-PAGE gels (10 μg protein/lane) and probed with specific antibodies. The following antibodies were utilized for western blot analysis: CD9 (1:2000; ab92726, Abcam), CD63 (1:1000; ab134045, Abcam), TSG101 (1:1000; sc-7964, Santa Cruz) and GM130 (1:1000; ab52649, Abcam). Anti-rabbit IgG or anti-mouse IgG, horseradish peroxidase (HRP)-linked antibody (1:2000; Cell Signaling Technology) was employed as a secondary antibody and chemiluminescent signals were detected using the ECL western Blot detection kit and Bio-RAD imaging system.

In the in vitro experiment, HMEC-1 cells were planted in a 6-well plate and treated with iMSCs-lEVs with or without U-46619. Control cells were cultivated for the same duration as the other groups, but without any treatment. Following that, protein samples from HMEC-1 cells were collected using RIPA lysis buffer (Beyotime, Jiangsu, China) and quantified with the Pierce BCA Protein Assay Kit (Cat#23225, Thermo Fisher Scientific). Cell lysates containing 10 μg protein were run on SDS-PAGE gels and probed with the following primary antibodies: anti-ERK (1:1000; 9102S, Cell Signaling Technology), anti-p-ERK (1:1000; 4370T, Cell Signaling Technology) and anti-β-actin (1:1000; ab133626, Abcam). The ECL western Blot detection kit and Bio-RAD imaging equipment were used to visualize the immunoreactive bands.

2.19. RNA-sequencing analysis

The RNA-seq analysis was performed by Shanghai Biotechnology Corporation (Shanghai, China). In brief, the total RNA of HMEC-1 cells was extracted using the RNeasy mini kit (Qiagen, Germany). The TruSeq™ RNA Sample Preparation Kit (Illumina, USA) was used to create paired-end libraries following the TruSeq™ Sample Preparation Guide.

2.20. Proteomic analysis

The iMSCs-lEVs proteomic analysis process was carried out by the Shanghai Applied Protein Technology Company (Shanghai, China). Identified iMSCs-lEVs proteins were compared with those from the Vesiclepedia database using FunRich Software (Version 3.1.3). FunRich Software (Version 3.1.3) was also used to perform gene ontology (GO) enrichment analyses. Ingenuity Pathway Analysis (IPA, Qiagen) was used to conduct a canonical pathway analysis.

2.21. Statistical analysis

GraphPad Prism 8 Software (CA, USA) was used for statistical analysis in this research. The Student's t-test was used to compare the two groups. One-way ANOVA was used to compare different groups. Two-way ANOVA was utilized to compare differences over time between three or more groups.

3. Results

3.1. Isolation & characterization of iMSCs-sEVs & iMSCs-lEVs

First, we applied flow cytometry to detect the surface markers of iMSCs. The results indicated that iMSCs highly expressed the positive markers of mesenchymal stem cells, including CD29, CD44, CD73, CD90, CD105 and CD146, while not expressing the negative markers CD34, CD45, CD133 and HLA-DR (Supplementary Figure S1). Next, we isolated and collected iMSC-secreted extracellular vesicles (sEVs) and large extracellular vesicles (lEVs) from the culture supernatant using differential centrifugation. Transmission electron microscopy (TEM) results showed that the diameter of isolated iMSCs-sEVs was approximately 100 nm, while iMSCs-lEVs had a diameter above 200 nm, both exhibiting cup-shaped structures (Figure 1A). Western blot analysis revealed that both iMSCs-sEVs and iMSCs-lEVs highly expressed markers CD9, CD63 and TSG101, but did not express the negative marker GM130 (Figure 1B). Nanoparticle tracking analysis (NTA) demonstrated that the majority of iMSCs-sEVs had a size distribution below 200 nm, whereas iMSCs-lEVs ranged from 0 to 600 nm (Figure 1C&D). Furthermore, we conducted a comprehensive analysis of various parameters pertaining to sEVs and lEVs. The particle count of iMSCs-lEVs in each milliliter of culture medium was determined to be 4.08 × 10⁷ ± 7.85 × 10⁶, while the particle count of iMSCs-sEVs in the same volume was 1.53 × 10⁸ ± 2.22 × 10⁷. Additionally, each iMSCs cell secreted an average of 3.40 × 10² ± 65.46 iMSCs-lEVs particles and 1.27 × 10³ ± 184.78 iMSCs-sEVs particles. In terms of protein content, each iMSCs-lEVs particle contained 2.26 × 10^-4 ± 4.63 × 10^-5ng of protein, while each iMSCs-sEVs particle contained 7.14 × 10^-5 ± 1.19 × 10^-5ng. Finally, the average protein content extracted from iMSCs-lEVs in each milliliter of culture medium was 8931.95 ± 254.08 ng, for iMSCs-sEVs was 10689.62 ± 285.24 ng (Figure 1E).

Figure 1.

Characterization of small extracellular vesicles and large extracellular vesicles derived from induced pluripotent stem cell-derived mesenchymal stem cells. (A) Representative image of lEVs and sEVs derived from iMSCs (iMSC-lEVs and iMSC-sEVs) scale bar = 100 nm. (B) Western blot showed positive exosome markers CD9, TSG101 and CD63 and negative marker GM130. (C & D) Size distribution of iMSC-sEVs and iMSC-lEVs measured by nanoflow cytometer. (E) Evaluation and quantification of iMSC-sEVs and lEVs.

CM: Conditioned medium; iMSC: Induced pluripotent stem cell-derived mesenchymal stem cell; iMSC-lEVs: Large extracellular vesicles from induced pluripotent stem cell-derived mesenchymal stem cell; iMSC-sEVs: Small extracellular vesicles from induced pluripotent stem cell-derived mesenchymal stem cell; lEVs: Large extracellular vesicles; sEVs: Small extracellular vesicles.

3.2. iMSCs-lEVs improve post-ischemic blood perfusion & vascular regeneration

Immediately after modeling, we injected iMSCs-sEVs (volume of 200ul, concentration of 1 × 10¹⁰/ml) and iMSCs-lEVs (volume of 200 ul, concentration of 1 × 10⁹/ml) at multiple sites in the quadriceps femoris, with an injection of the same volume of PBS serving as the control. Spectral imaging system analysis indicated that both types of vesicles resided around the femoral artery after injection (Supplementary Figure S2). To investigate whether iMSCs-lEVs could enhance blood flow perfusion in the ischemic hindlimbs of mice, we used a laser Doppler instrument to detect the degree of blood flow perfusion in mice on days 1, 7, 14 and 21 postoperation, with the contralateral right hindlimb serving as the control. The results showed that there was no significant change in blood flow in the hindlimbs of the sham-operated group. Starting from day 7 postoperation, the lEVs group exhibited significantly improved blood flow perfusion compared with the control group, with a trend of significant recovery in blood flow perfusion observed on days 14 and 21 postoperation. The interventional effect of the sEVs group was similar to that of the lEVs group, indicating that sEVs and lEVs have comparable therapeutic effects (Figure 2A&C). On day 21 postoperation, the results showed that the muscle cells in the sham-operated group were full and intact, with neatly arranged and symmetrical muscle fibers. In contrast, the muscle fibers in the control group became disorganized and exhibited atrophy. Compared with the control group, the iMSCs-sEVs and iMSCs-lEVs groups showed significantly reduced muscle fiber atrophy and more orderly and regular arrangement, indicating that iMSCs-lEVs have a certain protective effect on ischemic muscle cells (Figure 2B).

Figure 2.

Large extracellular vesicles from induced pluripotent stem cell-derived mesenchymal stem cells promoted mouse blood flow recovery and improved limb ischemia. (A) Laser Doppler blood flow imaging showed dynamic changes in blood perfusion in limb ischemia in each group on days 1, 7, 14 and 21 after iMSC-sEVs and iMSC-lEVs treatment (n = 5/group). (B) Representative images of HE staining after treatment with PBS, sEVs or lEVs, as well as sham group (n = 5/group). Scale bar: 100 μm. (C) Blood flow ratio of left limb (ischemic) to right limb (non-ischemic) in iMSCs-lEVs and iMSCs-sEVs treated mice. (D & E) The scores of ambulatory impairment and tissue damage after treatment in each group (n = 5/group). (F & G) Mouse endothelial cells maker CD31 and CD34 (n = 5/group). Scale bar: 100 μm. (H & I) Microvessel density in each group.

*p <0.05; **p <0.01; ***p <0.001; ^#p <0.0001.

Ctrl: control group; lEVs: Large extracellular vesicles; sEVs: Small extracellular vesicles; ns: Nonsignificant difference.

The analysis showed that the ambulatory impairment score and tissue damage score were significantly different between the iMSCs-lEVs group and the control group. On day 3, the limb functions of both the iMSCs-lEVs group and the control group showed a similar significant decline. On day 7, we observed the limb function gradually improved in the iMSCs-lEVs group. On day 21, the limb function of the sham group returned to normal levels, while the control group was more severe than other groups. Also, the ambulatory impairment score and issue damage score on the day 21 were significantly reduced. These data indicate that iMSCs-lEVs can promote functional recovery of ischemic limbs (Figure 2D & E).

The recovery of muscle following ischemia primarily relies on angiogenesis, as the formation of new blood vessels necessitates the provision of nutrients and oxygen. Therefore, in this study, we employed endothelial markers CD31 and CD34 to ascertain whether iMSCs-lEVs can stimulate angiogenesis in ischemic muscle. The results showed that on day 7, there was no significant difference in the number of neovascularization between the iMSCs-lEVs group and the iMSCs-sEVs group compared with the control group. However, on days 14 and 21, both the iMSCs-lEVs group and the iMSCs-sEVs group exhibited a significant increase in the number and prominence of neovascularization compared with day 7 within the same groups as well as the control group at all time points, indicating a notable angiogenic potential (Figure 2F–I). In summary, the intervention of iMSCs-lEVs can ameliorate the function of ischemic lower limbs through neovascularization.

3.3. iMSCs-lEVs promote migration, proliferation & tube formation of HMEC-1 cells

Endothelial cell migration, proliferation and tube formation are crucial processes in angiogenesis. However, the mechanisms underlying how iMSCs-lEVs promote angiogenesis remain elusive. We investigated the effects of iMSCs-lEVs on HMEC-1 function through a series of assays, including tube formation, transwell migration, CCK8 proliferation assay and wound healing experiments. The key to the function of iMSCs-lEVs lies in their internalization by cells. Therefore, labeled iMSCs-lEVs and iMSCs-sEVs with DiI dye were incubated with HMEC-1 cells. The results indicated that HMEC-1 cells successfully internalized these two types of vesicles (Supplementary Figure S3). In the tube formation assay, we observed that the control group exhibited limited tubular structure formation, whereas the lEVs group displayed prominent capillary-like structures (Figure 3A–C). In the wound healing assay, we monitored HMEC-1 cell migration at 6 and 12 h post-wounding. The data demonstrated that the endothelial cell migration capacity in the lEVs group was significantly higher than in the control group over time, with no significant difference between the lEVs and sEVs groups in their ability to promote cell migration (Figure 3D & F). Similar observations were made in the Transwell experiment following 24 h of iMSCs-lEVs and iMSCs-sEVs intervention (Figure 3E & G). Furthermore, through the CCK8 proliferation assay, we discovered that the effects of iMSCs-lEVs exhibited a dose-dependent relationship, with the effect of iMSCs-lEVs at a particle concentration of 10⁸/ml comparable to that of iMSCs-sEVs at a particle concentration of 10⁹/ml. Both 10⁸/ml iMSCs-lEVs and 10⁹/ml iMSCs-sEVs were capable of promoting the proliferation of human microvascular endothelial cells (Figure 3H).

Figure 3.

Large extracellular vesicles from induced pluripotent stem cell-derived mesenchymal stem cells promote migration, proliferation and tube formation of HMEC-1 cells in vitro. (A) Optical micrograph of tube formation assay under different group treatments (n = 3/group). Scale bar: 100 μm. (B & C) Quantitative analysis of total branch points and total capillary length (n = 3/group). Scale bar: 200 μm. (D & F) Optical micrographs of scratch wound assay at 6 and 12 h and quantitative analysis of migration index (n = 3/group). (E & G) Optical micrographs of Transwell assay (n = 3/group). Scale bar: 200 μm. (H) Cell proliferation rate under different group treatments (n = 3/group). (I & J) RT-qPCR analysis and ELISA of angiogenesis-related genes expression (n = 3/group).

*p <0.05; **p <0.01; ***p <0.001; ^#p <0.0001.

bFGF: Basic fibroblast growth factor; bFGFR: Basic fibroblast growth factor receptor; Ctrl: Control group; hbFGF: Human basic fibroblast growth factor; hEGF: Human epidermal growth factor; hVEGF: Human vascular endothelial growth factor; EGF: Epidermal growth factor; FGF: Fibroblast growth factor; lEVs: Large extracellular vesicles; ns: Nonsignificant difference; sEVs: Small extracellular vesicles; VEGF: Vascular endothelial growth factor.

sEVs can deliver proteins, lipids, RNAs and DNA from one cell to another, enabling intercellular communication, lEVs share some similar biological functions with sEVs. Therefore, we employed RT-qPCR and ELISA to detect the expression of angiogenesis-related genes, including FGF, EGF, VEGF and bFGF. Samples were collected from HMEC-1 cells after 48 h of lEVs intervention for RNA analysis. Compared with the control group, lEVs intervention significantly promoted the expression of these genes (Figure 3I & J). These results indicated that iMSCs-lEVs has the potential to promote angiogenesis.

3.4. RNA analysis of iMSCs-lEVs

Previous studies have indicated that the angiogenic capacity of endothelial cells is regulated by multiple genes, yet the specific molecular mechanisms remain elusive. Therefore, RNA-seq was applied to analyze the gene expression of HMEC-1 cells treated with lEVs. As a control, we used the gene expression profile of HMEC-1 cells following PBS intervention (control group). The gene heatmap revealed that, compared with the control group, 84 genes were upregulated (p <0.05) and 82 genes were downregulated in the lEVs group (p <0.05) (Figure 4A). Additionally, we further analyzed these transcriptomic data through gene set enrichment analysis (GSEA). The results showed that the lEVs group exhibited downregulation of gene sets associated with “suppression of angiogenic response in endothelial cells,” “suppression of proliferative response in endothelial cells” and “suppression of migratory capacity in endothelial cells” (Figure 4B–D). In other words, the lEVs group promoted the angiogenic, proliferative and migratory capabilities of endothelial cells. RT-qPCR randomly verified the characteristic genes that were significantly differentially expressed in the lEVs group (Figure 4E & F). Subsequently, we use Ingenuity Pathway Analysis (IPA) to analysis the RNA-seq, the results indicated that the differentially expressed genes were closely related to cell movement. Furthermore, they were highly associated with angiogenesis, including ‘IL-6 signaling pathway,’ ‘Wound Healing signaling pathway,’ ‘JAK/STAT3 signaling pathway,’ ‘EGF signaling pathway’ and ‘ERK/MAPK signaling pathway’ (Figure 4G–H). These results indicated that iMSCs-lEVs reprogramming the gene expression of HMEC-1 cells and modulating their angiogenic capacity.

Figure 4.

RNA-seq analysis of human microvascular endothelial cell line-1 cells transcriptome changes after Large extracellular vesicles from induced pluripotent stem cell-derived mesenchymal stem cells treatment. (A) Heatmap shows significantly differentially expressed genes in HMEC-1 treated with PBS and iMSC-lEVs. (B–D) GSEA analysis determine enrichment fractions of proliferation, differentiation and migration gene sets in HMEC-1 cells after iMSC-lEVs treatment. (E) The heatmap shows the differentially expressed genes related to proliferation, differentiation and migration in the PBS group and lEVs group in GSEA analysis. (F) RT-qPCR analysis of gene expression related to endothelial cell proliferation, differentiation and migration. (G) IPA shows disease and functional classification of differentially expressed genes in HMEC-1 cells. (H) IPA indicates canonical pathways of differentially expressed genes in HMEC-1 cells.

*p <0.05; **p <0.01; ***p <0.001; ^#p <0.0001.

Ctrl: control group; lEVs: Large extracellular vesicles; ns: Nonsignificant difference; sEVs: Small extracellular vesicles.

3.5. Proteomics analysis of iMSCs-lEVs

Recent studies have revealed that functional proteins encapsulated in extracellular vesicles play crucial roles in intercellular communication, signal transduction and various biological processes [22–24]. Through proteomic analysis, we identified 2408 proteins present in iMSCs-lEVs. Among them, 2222 proteins are included in the latest Vesiclepedia extracellular vesicle database, while 183 proteins remain unlisted (Figure 5A). Gene Ontology (GO) analysis of these proteins revealed that approximately 69.5% of the proteins are involved in metabolic processes, 18.6% participate in cell proliferation and those related to biological regulation also constitute a significant proportion during biological process analysis (Figure 5B). Cellular component analysis indicated that approximately 94.3% of the proteins originate from organelles (Figure 5C). Molecular function analysis demonstrated that proteins related to bridging and catalytic activities are significant components (Figure 5D).

Figure 5.

Proteomics analysis of iMSC-lEVs. (A) Venn diagram showing the overlapping and unique proteins in iMSC-lEVs with Vesiclepedia. (B) Gene ontology biological process analysis showed the proteins enriched in iMSC-lEVs. (C) Gene ontology cellular component analysis showed the proteins enriched in iMSC-lEVs. (D) Gene ontology molecular function analysis showed the proteins enriched in iMSC-lEVs. (E) IPA showed canonical pathway analysis of iMSC-lEVs proteins. (F & G) Western blot analysis showed p-ERK and ERK expression in HMEC-1 cells under different treatments (n = 3/group).

iMSC-lEVs: Large extracellular vesicles from induced pluripotent stem cell-derived mesenchymal stem cells; lEVs: Large extracellular vesicles.

3.6. iMSCs-lEVs regulate HMEC-1 partly by mediating the ERK/MAPK signaling pathway

To further explore whether iMSCs-lEVs mediate functional changes in downstream recipient cells through protein-mediated mechanisms, IPA was conducted on the proteins contained within iMSCs-lEVs. The data revealed that these proteins were associated with classical signaling pathways, including the ‘Actin Cytoskeleton Signaling Pathway,’ ‘mTOR Signaling Pathway,’ ‘ERK/MAPK Signaling Pathway’ and ‘HIF1α Signaling Pathway’ (Figure 5E). The data indicate that the biological functions of HMEC-1 cells are regulated by iMSCs-lEVs through key proteins, modulating their proliferative, migratory and angiogenic capabilities. Previous studies have demonstrated that the angiogenic capacity of vascular endothelial cells was regulated by multiple signaling pathways, including the VEGF signaling pathway, Notch signaling pathway, Angiopoietin-Tie signaling pathway, Wnt/β-catenin signaling pathway and ERK/MAPK signaling pathway [25–29]. As a result of combining previous transcriptomic gene expression profiling data, it was hypothesized that iMSCs-lEVs promote angiogenesis in endothelial cells by modulating the ERK/MAPK signaling pathway. To validate this hypothesis, HMEC-1 cells were cocultured with the ERK inhibitor SCH7772984 for 48 h [30]. Western blot demonstrated that the expression of p-ERK was partially reduced by the ERK inhibitor SCH7772984, whereas the expression of p-ERK was significantly upregulated in the iMSCs-lEVs group compared with the control group (Figure 5F & G). This indicated that the angiogenic capabilities of endothelial cells were enhanced by iMSCs-lEVs, at least partially through the activation of ERK phosphorylation (Figure 6).

Figure 6.

Schematic diagram of the therapeutic effect and mechanism of Large extracellular vesicles from induced pluripotent stem cell-derived mesenchymal stem cells in mouse hindlimb ischemia model. iMSC-lEVs treatment promotes angiogenesis by boosting endothelial cell proliferation, which is partially mediated by the ERK/MAPK signaling pathway.

iMSC: Induced pluripotent stem cell-derived mesenchymal stem cells; iMSC-lEVs: Large extracellular vesicles from induced pluripotent stem cell-derived mesenchymal stem cells.

4. Discussion

The core issue of critical limb ischemia (CLI) lies in the deficiency of perfusion to the muscles and tissues of the legs. Thus, enhancing the vascular regenerative capacity to improve perfusion in CLI is an important mechanism for treating the disease [31–33]. The process of angiogenesis involves endothelial cell sprouting from existing capillaries and migration and proliferation under the regulation of angiogenic factors such as VEGF and FGF produced by ischemic tissue, thereby forming new blood vessels. Therefore, protecting and repairing endothelial cell function is crucial in the treatment of lower limb ischemic diseases [34–36]. By improving the functional status of endothelial cells and promoting vascular regeneration and repair, the symptoms of lower limb ischemia can be effectively alleviated [37]. In this study, we intervened in HMEC-1 cells with iMSCs-lEVs and found that iMSCs-lEVs significantly promoted the proliferation, migration and tube-forming ability of these cells.

In addition to size, small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs) also differ in biological characteristics and composition. sEVs are typically enriched with specific protein markers (e.g., CD9, CD63, TSG101), while lEVs may contain more cellular membrane, organelle and protein components. Previous studies have indicated that sEVs can promote angiogenesis, neurogenesis and regulate autophagy processes, thus aiding in the repair of ischemic injury [38–40]. Additionally, sEVs may possess direct anti-inflammatory and immunomodulatory effects, mitigating ischemic damage and reducing local or systemic inflammatory responses [41–43]. These effects contribute to alleviating tissue injury and creating a more favorable environment for the regeneration and repair of damaged cells. While previous research has predominantly focused on sEVs, recent studies on lEVs have increased, demonstrating that lEVs may play important roles in intercellular signaling and regulation. In this study, we found that lEVs significantly improved blood flow perfusion in ischemic mouse limbs.

In the selection of vesicle sources, we opted for human induced pluripotent stem cells (iPSCs) as the source of stem cells. Compared with adult mesenchymal stem cells (MSCs), iPSCs can be cultured for dozens of generations in vitro while maintaining good proliferation and differentiation capabilities. Moreover, the technology for reprogramming adult iMSCs into iPSCs is relatively mature [44–46]. Our previous research has successfully differentiated iPSCs into iMSCs and applied them in a mouse model of cerebral ischemia, achieving favorable therapeutic effects [47]. In this study, we found that iMSCs-lEVs can promote the proliferation, migration and tube formation of endothelial cells to improve blood flow perfusion in the ischemic limbs. The extraction process of lEVs is more convenient than that of sEVs, suggesting that lEVs may have better prospects for treating ischemic injuries.

Our study also investigate the comparative yield and therapeutic effects of sEVs and lEVs isolated from the same volume of iMSCs culture medium. Both our previous research and the current study have confirmed that, at least in modulating macrophage polarization to alleviate tendon pain and promoting endothelial cell proliferation to restore blood flow perfusion in lower limb ischemia, lEVs at a concentration of 1 × 10⁹ particles/ml can achieve similar functionality to 1 × 10¹⁰ particles/ml sEVs. This may be attributed to the possibility that individual lEVs encapsulate more functional molecules than sEVs [48].

The RNA sequencing results revealed differential gene expression in endothelial cells under lEVs intervention, with significant upregulation observed in signaling pathways such as cellular movement, cell death and survival, and cellular growth and proliferation. Increasing evidence have reported that the ERK/MAPK signing pathway plays a crucial role in promoting the regulation of endothelial cell migration, proliferation and vascular regeneration. Wang et al. found that mifepristone enhances angiogenesis and cell permeability through the ERK MAPK pathway (rather than the p38 and JNK signing pathways) [42]. Mavria et al. demonstrated that ERK-MAPK promotes endothelial cell survival and sprouting by downregulating Rho kinase signaling [49]. Our data suggested that lEVs promoted vascular regeneration, at least through the ERK/MAPK pathway. However, the proteins transferred by iMSCs-lEVs require further data comparison and relevant molecular experiments for validation. In addition, in this study, we found that the proteins contained in iMSCs-lEVs also participate in the ‘Actin Cytoskeleton signaling pathway,’ ‘mTOR signaling pathway,’ ‘ERK/MAPK signaling pathway’ and ‘HIF1α signaling pathway.’ Whether other pathways are involved in the angiogenic process of endothelial cells and whether these pathways cooperate remain to be further explored in future experiments.

In summary, we have identified a method for the rapid isolation of lEVs compared with sEVs, with similar therapeutic effects, at least in promoting recovery in a lower limb ischemia model. lEVs regulated the ERK/MAPK signing pathway, promoting endothelial cell proliferation, tube formation and migration to restore blood supply to the lower limbs. These findings provided a novel therapeutic approach for the treatment of lower limb ischemic diseases.

5. Conclusion

Our study found that iMSC-lEVs can effectively promote the recovery of blood flow perfusion in mouse lower limb ischemic tissues and improve their function after lower limb ischemia. In addition, iMSC-lEVs can increase endothelial cell proliferation, differentiation and angiogenic ability by regulating the ERK/MAPK signing pathway. These findings revealed the important role of iMSC-lEVs in angiogenesis and provided a novel cell-free therapy for improving lower limb ischemic injury and other ischemic diseases.

Funding Statement

This work was supported by the National Natural Science Foundation of China (Grant No. 81870972).

Supplemental material

Supplementary data for this article can be accessed at https://doi.org/10.1080/17435889.2024.2363743

Author contributions

Z Huang: conceptualization, investigation, writing – original draft. Z Chen: investigation, writing – original draft. T Ye: writing – original draft, data curation. L Luo: investigation. J Zhang: investigation. Q Li: conceptualization, writing – review & editing. Y Wang: conceptualization, writing – review. B Zhao: funding acquisition.

Financial disclosure

This work was supported by the National Natural Science Foundation of China (Grant No. 81870972). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

All animal experimental procedures were approved by the Animal Research Committee of Shanghai Jiao Tong University Affiliated Sixth People's Hospital (SYXK2021-0028, Shanghai, China).