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Published in final edited form as: Brain Hemorrhages. 2020 Nov 30;2(1):57–62. doi: 10.1016/j.hest.2020.10.007

Therapeutic effects of exosomes from angiotensin-converting enzyme 2 - overexpressed endothelial progenitor cells on intracerebral hemorrhagic stroke

Jinju Wang a, Shuzhen Chen a, Sri Meghana Yerrapragada a, Wenfeng Zhang a, Ji C Bihl a,b,*
PMCID: PMC11932701  NIHMSID: NIHMS2049628  PMID: 40129927

Abstract

Objective:

We have previously demonstrated that angiotensin-converting enzyme 2 (ACE2) could boost the therapeutic effects of endothelial progenitor cells (ACE2-EPCs) on stroke. However, where this effect comes from is still unclear. Here, we investigated whether the exosomes (EXs) released from ACE2-EPCs could provide the benefit for acute intracerebral hemorrhagic stroke (ICH).

Methods:

The C57BL/6 mice were induced ICH by collagenase injection, followed by intravenously administration of ACE2-EPC-EXs. ACE2 blocker, DX600 was used to verify the effects of ACE2. The neurological deficit score (NDS), hemorrhage volume, brain water content, and blood–brain barrier (BBB) permeability were measured at day 2 after injection. The levels of ACE2 and inflammatory factors/genes in the brain were also measured.

Results:

EPC-EXs decreased hemorrhage volume, brain edema, BBB permeability, and improved NDS, which were enhanced by ACE2-EPC-EXs treatment; 2) As compared to EPC-EXs, ACE2-EPC-EXs resulted in an up-regulation of ACE2 in the brain, associating with the down-regulated expressions of TNF-α and NFκB and up-regulated level of IκBα. 3) DX600 blocked the above mentioned protective effects of ACE2-EPC-EXs in ICH mice.

Conclusion:

These data suggest that the infusion of ACE2-EPC-EXs could provide the therapeutic effect on acute ICH by alleviating the post-stroke inflammation via transferring ACE2.

Keywords: Intracerebral hemorrhage stroke, Angiotensin-converting enzyme 2 (ACE2), Exosomes (EXs), Endothelial progenitor cells (EPCs)

1. Introduction

Intracerebral hemorrhagic stroke (ICH) is a subtype of hemorrhagic stroke with high morbidity and mortality. Primary ICH is caused by small arteriole ruptures which stem from vascular pathological changes.1 The vascular pathologies including intimal thickening, plaque formation, and vascular remodeling are vulnerable to vessel ruptures. After vessel rupture, the hemoglobin enters brain tissue and induces a series of consequential pathological changes, such as edema, inflammation, cell death, which ultimately cause neurological deficits.2 Early inflammatory reactions in ICH include accumulation of the inflammatory substance such as tumor necrosis factor- α (TNF-α) released by inflammatory cells such as microglia.3 The nuclear factor-kappa B (NF-kB) inflammatory signal pathway has been shown to be activated in ICH4,5 and the production of inflammatory cytokines was increased in the acute phase of ICH in the brain.6,7 Hence, inhibition of inflammation might be a promising therapeutic strategy for treating ICH.

Endothelial progenitor cells (EPCs), progenitors of endothelial cells, are well-known to play critical roles in maintaining vascular homeostasis and participating in endothelial repair.8 Previous studies have indicated that EPCs could reduce tissue injury, promote angiogenic repair, and functional recovery in ischemic brain or limb.9,10 Our previous studies have shown the therapeutic effects of EPCs on ischemic stroke.11 Of note, we found that these beneficial effects of EPCs are from their released exosomes.32,33 It is known that exosomes (EXs; diameter: 30–120 nm) are a type of extracellular vesicles that could be released by all types of cells. A large body of studies has demonstrated that EXs can mediate cell–cell/tissue communication mainly via conveying their cargoes such as proteins and microRNAs.12 Indeed, inducible pluripotent stem cell EXs have been shown to elicit protective effects on cardiomyocytes against ischemic injury.13 Ma and colleagues have revealed that EXs derived from EPCs (EPC-EXs) can protect brain cells against hypoxic injury.14 Recently, we found that EPC-EXs could provide protective effects on oxyhemoglobin-induced neuron death in vitro.33 Nevertheless, there is limited information regarding the effects of EPC-EXs on ICH.

Angiotensin-converting enzyme 2 (ACE2), a homolog of ACE, is abundantly expressed in the cardiovascular-related areas of the brain and blood vessels. An earlier study showed that pharmacological activation of ACE2 induced beneficial effects in ischemic stroke.15 Human umbilical cord mesenchymal stem cells-combined with ACE2 has been shown to exhibit a better effect than ACE2 alone on alleviating acute lung ischemia–reperfusion injury.16 We have demonstrated that neuronal overexpression of ACE2 protects the brain from ischemia-induced damage.11 Infusion of EPCs over-expressed ACE2 (ACE2-EPCs) enhanced the efficacy of EPC-based therapy for ischemic stroke.11 Most recently, we discovered that their released exosomes could be one of the underlying mechanisms. Exosomes from ACE2-EPCs (ACE2-EPC-EXs) have been shown to enhance the protective effect of EPC-EXs on ageing and hypoxia/reoxygenation-injured endothelial cells (ECs) by promoting the survival and function of ECs.17,18 Increasing studies recognize the role of ACE2 in regulating the inflammatory process in various disease conditions such as cardiac hypertrophy, pulmonary hypertension.19 ACE2 has been shown to inhibit the NF-kB pathway to attenuate inflammatory response in hyperoxic lung injury.20 We were wondering whether ACE2 could enhance the effects of EPC-EXs on ICH in the acute phase by alleviating the inflammation.

In this study, we determined the therapeutic effects of ACE2-EPC-EXs on acute brain injury of ICH mice and whether the inflammatory pathway is involved.

2. Materials and methods

2.1. Animals

C57BL/6 mice (8–10 weeks of age; n = 10/group) were used in this study. All mice were maintained in a 22 °C room with a 12-hr light/dark cycle and fed with standard chow and drinking water ad libitum. All experimental procedures were approved by the Wright State University Laboratory Animal Care and Use Committee and were following with the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health (NIH).

2.2. Microinjection of collagenase to induce ICH model

The collagenase injection surgery was conducted using a mouse stereotaxic frame as we previously reported.21 Ketamine: Xylazine mixture (100:8 mg/kg, 20–30 μl, i.p) was used for anesthesia. A 1.0-mm burr hole was made on the skull by using a dental drilltrephine. Collagenase (type VII-S; 0.075 U/0.5 μl) was injected into the caudate-putamen by injection cannula (a glass micro-needle, 30 lm in diameter) at a rate of 0.5 μl/min. The cannula is localized using stereotaxic coordinates related to bregma (0.5 mm anterior to bregma, 3.0 mm right lateral to the midline, 4.0 mm in depth). In sham mice, a 1.0 mm burr hole was made on the skull, but no collagenase was administrated.

2.3. Overexpression of ACE2 on EPCs

Human EPCs (Celprogen, Torrance, CA) were cultured in complete growth medium (Celprogen; Torrance, CA). EPCs were transfected with lentivirus (lenti)-GFP or lenti-GFP-ACE2 cDNA (10 nM, Applied Biological Materials Inc., Richmond, Canada) for 48 hrs to generate the null-EPCs and ACE-EPCs.2 Transduction efficiency [the percentage of the green fluorescent protein (GFP)-expressing cells] was quantified by direct counting under a fluorescence microscope (EVOS; Thermo Fisher Scientific).

2.4. Preparation of EPC-EXs and ACE-EPC-EXs

To stimulate the generation of EXs, EPCs and ACE2-EPCs were cultured with serum-free conditional medium (Celprogen; Torrance, CA) for 24 hrs. Then, the conditional medium was collected and used for EX isolation.22 In brief, the medium was centrifuged at 300 g for 15 mins followed by 2000g for 30 mins and 20,000g for 70 mins. Then the supernatant was ultracentrifuged at 170,000 g for 90 mins. The EPC-EXs and ACE2-EPC-EXs were isolated from the culture medium of null-EPCs and ACE-EPCs, respectively.

2.5. Treatment groups

Two hours after collagenase-induced ICH, mice were randomly divided into four groups: vehicle (administrated with 100 μl PBS only), EPC-EXs (1 × 1011 EXs/100 μl), ACE2-EPC-EXs (1 × 1011 EXs/100 μl), EPC-EXs (1 × 1011 EXs/100 μl) + DX600 (0.1 μM/kg, i.v). The dose of DX600 (Cayman Chemical) was based on a previous report.23 EXs were transfused via tail vein injection. The dose of EXs was based on a previous study.24 Pain and discomfort were minimized by an initial injection of Buprenorphine (0.1 mg/kg, s.c) and carprofen (5 mg/kg, s.c) followed with another carprofen injection within 24 hrs.

2.6. Neurological deficit score (NDS) evaluation

Two days after the EX treatment, the NDS of all mice was determined by using a 24-point scoring system,25 including body symmetry, gait, climbing, circling behavior, front limb symmetry, and compulsory circling. Each test was graded from 0 to 4, establishing a maximum deficit score of 24. All behavioral tests were conducted in a quiet and lowlight room by observers who were blind to the treatment condition.

2.7. Measurement of hemorrhagic size

The analysis of the hemorrhage volume was performed per our previous publication.21 On day 2, the brain from different experimental groups was collected and cut into coronal slices (1 mm thickness). Images of the brain slices were then taken with a digital camera. Hematoma size was calculated by ImageJ software (NIH, MD, USA). Damaged volume in one brain was estimated from the sum of all value calculated from the formula: [(area of the damaged region in each section) × 1] (mm3).

2.8. Brain water content

Brain water content was evaluated via the wet-dry weight method, as previously described.26 Briefly, mice were sacrificed on day 2 after ICH, brains were immediately removed, weighed, and cut into 4 mm thick sections. Then the brain sections were dried at 100 °C for 24 hrs to determine the dry weight. Brain water content was calculated as (wet weight – dry weight)/wet weight × 100%.

2.9. BBB permeability analysis

On day 2 after EX treatment, BBB permeability was assessed by Evan’s blue dye as previously reported.27 In brief, 4% Evan’s blue dye (Sigma-Aldrich, MO, USA) was slowly injected into the tail vein. Three hours later, mice were sacrificed, and the brains were quickly removed and sliced into 1 mm-thick coronal sections. The injured brain tissue was dyed blue. To measure the amount of extravasated Evan’s blue, brain samples were weighed, homogenized in 50% trichloroacetic acid (TCA), and centrifuged. The supernatant was mixed with 4-fold ethanol, then incubated at 4 °C overnight. Then the supernatant was collected and spectrophotometrically quantified at 620 nm. Data were expressed as mean ± SE after normalization with the sham group.

2.10. ELISA assay

The proteins were extracted from the ICH brain with lysis buffer (Thermo Scientific, FL) containing protease inhibitors cocktail (Sigma). The samples were evaluated in duplicates. The levels of ACE2 and TNF-a in the brain were determined by ELISA kits (R&D System, Minneapolis, MN) according to the manufacturer’s instructions.

2.11. Western blot analysis

The proteins of the brain samples were subjected to electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked by incubating with 5% dry milk for 1 hr and then incubated with primary antibody against-NFkB (1:1000; Cell signaling) or IkBa (1:1000; Cell signaling) at 4 °C overnight. After being washed thoroughly, membranes were incubated with horseradish peroxidase (HRP) conjugated IgG (1:40000; Jackson ImmunoResearch Labs, INC. PA) for 1 hr at room temperature. Blots were then developed with enhanced chemiluminescence developing solutions and quantified. β-actin (1:4000; Sigma-Aldrich, St. Louis, MO) was used to normalize protein loading.

2.12. Statistical analysis

All data, excepting neurologic deficit scores, are presented as mean ± SE. The neurologic deficit scores were expressed as median (range). Multiple comparisons were analyzed by two-way ANOVA (SPSS version 16.0; SPSS, Chicago, IL, USA) followed by the Tukey test. For all tests, a P-value < 0.05 was considered significant.

3. Results

3.1. EPC-EXs reduced hemorrhage volume and improved the neurological deficit in acute ICH, which was enhanced by ACE2-EPC-EXs

First of all, we confirmed the transfection efficacy by checking the number of green cells (GFP-expressing cells) after transfection. The data showed that the transduction efficiency was about 97 ± 2% (Sup Fig. 1.). To evaluate the hematoma size, the brain samples were collected and imaged on day 2 after treatments. As shown in Fig. 1A, the sham group has no hemorrhage in the brain, while in the vehicle group we can see some hemorrhage in different brain sections indicating the success of the ICH animal model. The summarized data showed that (Fig. 1B) the non-treated mice (vehicle) had a hemorrhage volume of 13.2 ± 1.8 mm3, while the mice treated with EPC-EXs had a smaller hemorrhage volume (9.1 ± 1 mm3) (P < 0.05). The ACE2-EPC-EXs treated mice had the smallest hemorrhage volume (6 ± 0.5 mm3). ACE2 inhibitor DX600 partially blocked the effects elicited by ACE2-EPC-EXs. This suggests that ACE2-EPC-EXs could boost the effects of EPC-EXs on limiting the hemorrhage volume thought the effects of ACE2.

Fig. 1.

Fig. 1.

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Analyses of NDS and hemorrhagic size in ICH mice on day 2 after treatment. A. Representative images of hemorrhage in the brain. B. Summarized data showing the hemorrhage volume in each group. C. Summarized data of NDS in each group. *P < 0.05 vs. vehicle, +P < 0.05 vs. EPC-EXs, #P < 0.05 vs. ACE2-EPC-EXs, n = 10/group. Data are mean ± S.E. NDS: neurological deficit score.

As shown in Fig. 1C, the mice in the vehicle group (PBS infusion group) had a higher NDS than the ones in the sham group indicating the neurological deficits of mice after ICH induction. The mice received EPC-EXs treatment had a lower NDS than that in the vehicle group on day 2 (P < 0.05). Those administrated with ACE2-EPC-EXs had an even lower NDS as compared to the ones treated with EPC-EXs (P < 0.05). These data suggest that EPC-EXs could improve the neurological function as early as day 2, meanwhile, ACE2-EPC-EXs could enhance the beneficial effect. Of note, DX600 significantly raised NDS when compared to the ACE2-EPC-EXs group, indicating that the beneficial effect is from the ACE2.

3.2. ACE2-EPC-EXs had better effects than EPC-EXs on alleviating brain edema and improving BBB permeability in acute ICH

As revealed by the brain water content analysis (Fig. 2A), vehicle mice had a higher water content than that in sham mice (83.2 ± 0.7 % vs. 78.5 ± 0.4 %, vehicle vs. sham, P < 0.05). ACE2-EPC-EXs had a better effect than EPC-EXs on alleviating brain edema as revealed by decreased brain water content (80.6 ± 0.2 % vs. 81.6 ± 0.3 %, ACE2-EPC-EXs vs. EPC-EXs, P < 0.05). The BBB permeability was increased in the vehicle mice (1 ± 0.2). EPC-EXs treatment reduced the BBB permeability (0.82 ± 0.4), which was further alleviated by ACE2-EPC-EXs (0.75 ± 0.2, P < 0.05). Similarly, DX600 partially blocked the effects of ACE2-EPC-EXs.

Fig. 2.

Fig. 2.

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Analyses of brain edema and BBB permeability in ICH mice on day 2 after treatment. A. summarized data showing the brain WC in each group. B. BBB permeability in each group. *P < 0.05 vs. vehicle, +P < 0.05 vs. EPC-EXs, #P < 0.05 vs. ACE2-EPC-EXs, n = 10/group. Data are mean ± S.E. WC: water content; BBB: the blood–brain barrier.

3.3. ACE2-EPC-EXs raised ACE2 level in the brain of acute ICH mice

To further understand the mechanism of the beneficial effects induced by ACE2-EPC-EXs, we analyzed the protein levels of ACE2 in the brain of ICH mice. Our results (Fig. 3A) showed that EPC-EXs infusion did not affect the expression of ACE2 in the brain as compared to that in the sham or vehicle group, but ACE2-EPC-EXs treatment significantly raised ACE2 level in ICH mice (P < 0.05). DX600 did not significantly change the expression of ACE2.

Fig. 3.

Fig. 3.

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Analyses of ACE2 and TNF-α levels in the brain of acute ICH mice. A. the level of ACE2 in each group. B. the level of TNF-α in the ipsilateral brain. *P < 0.05 vs. vehicle, +P < 0.05 vs. EPC-EXs, #P < 0.05 vs. ACE2-EPC-EXs, n = 10/group. TNF-α: tumor necrosis factor.

3.4. ACE2-EPC-EXs downregulated the expressions of TNF-α and NFκB, whereas upregulated IκBα level in the brain of acute ICH mice

As inflammation is a very important player of the pathophysiological changes in the acute phase of ICH, we detected the level of inflammatory pathway. The level of pro-inflammatory cytokines such as TNF-α was measured by ELISA assay. We found that (Fig. 3B) there was an increased level of TNF-α in the vehicle mice when compared to the sham ones (1 ± 0.03 vs. 0.43 ± 0.03, vehicle vs. sham, P < 0.05). EPC-EXs treatment decreased TNF-α production (0.76 ± 0.06) in the brain tissue, which was further reduced by ACE2-EPC-EXs (0.5 ± 0.04). Meanwhile, our data (Fig. 4) showed that the gene expressions of NFκB were downregulated whereas the IκBα level was upregulated by EPC-EXs. ACE2-EPC-EXs elicited even better effects. DX600 reduced the downregulation of TNF-α and NFκB as well as upregulated expression of IκBα.

Fig. 4.

Fig. 4.

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Analysis of the gene expression of NFκB and IκBα in the brain of acute ICH mice. A. the expression of NFκB in the ipsilateral brain tissue. B. the expression of IκBα in the ipsilateral brain tissue. *P < 0.05 vs. vehicle, +P < 0.05 vs. EPC-EXs, #P < 0.05 vs. ACE2-EPC-EXs, n = 10/group. Data are mean ± S.E.

4. Discussion

In the present study, we have demonstrated that EPC-EX infusion exhibited protective effects on collagenase-induced ICH in the acute phase. What’s more, all of these effects were boosted by ACE2, which was verified by the application of the ACE2 inhibitor DX600.

EXs released from EPCs have generated great interest based on their ability to alleviate inflammation in the lung,28 reduce tissue injury in the heart, promote angiogenic repair and functional recovery in the brain and limb.9,10 In the present study, we focused on investigating the effects of EPC-EXs infusion on acute brain injury in a collagenase-induced ICH mouse model. First of all, our data showed that EPC-EXs treatment alleviated the functional neurological deficits, reduced the hematoma size and brain edema on day 2 after ICH onset. Edema is one of the main pathophysiological features in ICH, and the main cause of edema is because of the damages of the BBB.29 Indeed, we found that there was increased BBB permeability in the vehicle mice. Whereas EPC-EXs infusion alleviated the BBB permeability as evidenced by less Evan’s blue extravasation into the brain tissue. What’s more, our data revealed that ACE2 could enhance the efficacy of EPC-EXs. We found that there was a higher level of ACE2 in the ipsilateral brain of ICH mice. This might be ascribed to the ability of EPC-EXs which can convey their carried ACE2 to recipient cells.17 These findings are supported by previous studies showing that ACE2 could boost the beneficial effects of mesenchymal stem cells and EPCs on acute lung ischemia–reperfusion injury16 or ischemic stroke animal models.30 As we have known, the key factor that affects the ICH outcome is hemorrhagic volume. The management of acute ICH including removal of the blood clot, controlling of the hematoma expansion, and decreasing of brain edema is very important for a better outcome. However, the hematoma itself can lead to secondary brain injury resulting in severe neurological deficits and sometimes delayed fatality. To better understand the mechanisms that trigger the pathophysiological changes in and around the intracerebral hematoma are very helpful for exploring novel therapeutic avenues for ICH.

Dysregulated inflammation is a complicated pathological process involved in various diseases, and the treatment of inflammation-linked disorders currently represents an enormous global burden. Inflammation plays a prominent role in the pathogenesis of ICH. An inflammatory response in the surrounding brain occurs soon after ICH and peaks several days later in human beings and in animals. Excessive production of inflammatory factors such as TNF-α could result in tissue injury in ICH.31 Several studies have demonstrated that the NF-κB inflammatory signal pathway is activated in ICH.4,5 EPCs have been shown to modulate inflammation-associated ischemic stroke vasculome.4,5 In the present study, we studied whether EPC-EXs could target the NF-κB signal pathway to alleviate the inflammatory response. As expected, our data showed that mice receiving EPC-EXs had a low level of TNF-α in the ipsilateral brain as revealed by ELISA analysis. Meanwhile, NF-kB expression was down-regulated and IκBα level was upregulated. What’s more, ACE2-EPC-EX treatment elicited even better effects. The boosted therapeutic effects of ACE2-EPC-EXs is largely ascribed to their carried ACE2. This assumption was confirmed by the application of ACE2 inhibitor DX600 in vivo. Most importantly, the data of attenuated inflammatory gene expression was consistent with the reduced BBB permeability and improved NDS. As inflammation aggravates hemorrhagic brain injury, the decreased inflammation could be one of the mechanisms that EPC-EXs and ACE2-EPC-EXs benefit the ICH. The present study provided evidence that the EPC-EXs and ACE2 are involved in alleviating early brain injury and promoting neurological functional repair after ICH via decreasing the inflammation pathway, which could provide a potential therapeutic strategy for ICH. Although it is unclear whether the improvement of NDS is due to the promoted angiogenesis or neurogenesis, our previous study has shown that infusion of EPC-EXs could promote the angiogenesis or neurogenesis in ischemic stroke mice,32 and overexpressing ACE2 in EPCs could enhance the beneficial effect of EPC-based therapy for ischemic stroke.30 Future studies are warranted to determine the location of transfused EPC-EXs by analyzing the dynamics of transfused EPC-EXs in living animals, as well as the underlying mechanisms of the beneficial effects of ACE2-EPC-EXs in improving neurological dysfunctions.

Taken together, in the current research, we have demonstrated for the first time that mice receiving EPC-EXs as a therapeutic strategy showed alleviated brain injury, which was enhanced by ACE2-EPC-EXs. The privilege of using ACE2-EPC-EXs for treating ICH is that ACE2-EPC-EXs might pass the ACE2 to local brain cells such as endothelial cells and neurons, future work will be conducted regarding this.

Supplementary Material

Supplementary data

Acknowledgment

This work was supported by the National Institute of Neurological Disorders and Stroke (1R01NS102720), and the American Heart Association (16SDG26420078).

Footnotes

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.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.hest.2020.10.007.

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