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. Author manuscript; available in PMC: 2017 Feb 1.
Published in final edited form as: Stem Cells. 2016 Jul 8;34(12):2943–2955. doi: 10.1002/stem.2448

Human Mesenchymal Stem Cell-Derived Microvesicles Prevent the Rupture of Intracranial Aneurysm in Part by Suppression of Mast Cell Activation via a PGE2-Dependent Mechanism

Jia Liu 1, Atsushi Kuwabara 1, Yoshinobu Kamio 1, Shuling Hu 1, Jeonghyun Park 1, Tomoki Hashimoto 1, Jae-Woo Lee 1
PMCID: PMC5287411  NIHMSID: NIHMS845293  PMID: 27350036

Abstract

Background

Activation of mast cells participates in the chronic inflammation associated with cerebral arteries in intracranial aneurysm formation and rupture. Several studies have shown that the anti-inflammatory effect of mesenchymal stem cells (MSCs) is beneficial for the treatment of aneurysms. However, some long-term safety concerns exist regarding stem cell-based therapy for clinical use.

Objective

We investigated the therapeutic potential of microvesicles (MVs) derived from human MSCs, anuclear membrane bound fragments with reparative properties, in preventing the rupture of intracranial aneurysm in mice, particularly in the effect of MVs on mast cell activation.

Methods and Results

Intracranial aneurysm was induced in C57BL/6 mice by the combination of systemic hypertension and intrathecal elastase injection. Intravenous administration of MSC-derived MVs on day 6 and day 9 after aneurysm induction significantly reduced the aneurysmal rupture rate, which was associated with reduced number of activated mast cells in the brain. A23187-induced activation of both primary cultures of murine mast cells and a human mast cell line, LAD2, was suppressed by MVs treatment, leading to a decrease in cytokine release and tryptase and chymase activities. Up-regulation of prostaglandin E2 (PGE2) production and E-prostanoid 4 (EP4) receptor expression were also observed on mast cells with MVs treatment. Administration of an EP4 antagonist with the MVs eliminated the protective effect of MVs against the aneurysmal rupture in vivo.

Conclusions

Human MSC-derived MVs prevented the rupture of intracranial aneurysm, in part due to their anti-inflammatory effect on mast cells, which was mediated by PGE2 production and EP4 activation.

Keywords: intracranial aneurysm, mast cells, mesenchymal stem cells, microvesicles, prostaglandin E2

INTRODUCTION

The spontaneous rupture of intracranial aneurysms is a significant source of morbidity and mortality in patients. Surgical clipping and/or endovascular coiling are the primary clinical treatment options to prevent future aneurysmal rupture. However, the development of nonsurgical innovative therapies is needed for patients at high risk for surgery or with inoperable aneurysms. Recent studies have found that inflammation plays a critical role in the pathogenesis of aneurysm formation and rupture. An increased number of inflammatory cells including mast cells can infiltrate into the aneurysmal walls and participate in the inflammatory response by releasing a wide range of mediators, such as cytokines, chemokines and proteases, which promote vascular destruction [1–4]. Immunomodulatory therapies may provide a potential, non-surgical intervention to prevent the rupture of intracranial aneurysm.

In various models of organ injury, mesenchymal stem cells (MSCs) have been found to release microvesicles (MVs), which were biologically active as the parent stem cells as a therapeutic with immunomodulatory properties [5, 6]. MVs are anuclear membrane bound fragments, 50–200 nm in size, constitutively released from live cells as exosomes from the endosomal compartment or as shedding vesicles from the plasma membrane [7]. Similar to their parent cells, MVs derived from MSCs selectively accumulate at the lesion sites, where they mediate the processes of tissue repair and anti-inflammation through the transfer of proteins, lipids and RNA into target cells [5, 6]. Our group previously reported that human MSC-derived MVs attenuated alveolar inflammation in a mouse model of acute lung injury in part through the up-regulation of keratinocyte growth factor mRNA [8].

In the current study, we hypothesized that MSC-derived MVs can prevent aneurysmal rupture in a mouse model of intracranial aneurysm [9, 10] in part through the immunomodulatory effect of MVs on mast cells. To further investigate the underlying mechanism, we noted that prostaglandin E2 (PGE2) plays a key role in regulating the activation, maturation and cytokine secretion of many immune cells [11]. In addition, PGE2 can act as an important contributor to the resolution of inflammation in particular through the activation of EP4 [12, 13]. We therefore aimed to study if PGE2 and activation of its receptor were involved in the immunomodulation of MSC-MVs on mast cell activity.

MATERIALS AND METHODS

An expanded Materials and Methods section is available in the Online Supplements.

MSCs Culture and Isolation of MSC-Derived Microvesicles

Human bone marrow-derived MSCs were obtained from a National Institutes of Health repository in the Texas A & M Health Science Center (Temple, TX) and cultured as previously described [8]. Cells at passages 3–10 were used for experiments and for microvesicle isolation. The viability of human MSCs prior to MVs isolation was measured as >95% by trypan blue exclusion, excluding apoptotic bodies mixed in with the released MVs. MVs were obtained from the supernatants of serum-deprived MSCs, using ultracentrifugation at 100,000 g for 1 h at 4°C twice, as previously described [8]. Isolated MVs were resuspended in phosphate buffered saline (PBS) according to the final cell count of MSCs (10 μL per 1 × 106 cells) and stored at −80°C prior to use.

Mast Cells

Bone marrow-derived mast cells (BMMCs) were isolated from mice and maintained in culture as described in the Online Supplements. BMMCs, after 6–8 weeks of culture, were used for experiments only when > 95% were identified as mast cells based on the presence of metachromatic granules and cell surface expression of CD117 and FcεR-1α, as determined by toluidine blue staining and flow cytometry analyses respectively. The human mast cell line LAD2 was kindly provided by Dr. Arnold Kirshenbaum in the National Institute of Allergy and Infection Diseases and maintained as previously described [14].

Assessment of PKH26-Labeled MVs Internalization into BMMCs

MVs were labeled with red fluorescent dye PKH26 according to manufacturer’s protocol (Sigma-Aldrich, Ann Arbor, MI). PKH26-labeled MVs, pretreated with or without anti-CD44 neutralizing antibody, were incubated with BMMCs over 15 h, followed by analysis on BD™ LSR II flow cytometry with FACSDiva software (BD Biosciences, San Jose, CA) or under a Zeiss LSM700 confocal microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY). As a control for non-specific labeling of the cells, PKH26 dye was added to PBS without MVs, centrifuged and washed (indicated as “PKH26-PBS”) and incubated with BMMCs.

Intracranial Aneurysm Model and MVs Administration

Intracranial aneurysms were induced in nine-week-old male mice (C57BL/6 mice, 20–25 gms, Jackson Laboratory) as previously described with minor modifications [9, 10]. All animal procedures were approved by the Institutional Animal Care and Use Committee at UCSF. Briefly, aneurysm induction was performed by combining a single injection of elastase into the cerebrospinal fluid and deoxycorticosterone acetate (DOCA)-salt hypertension [15]. Aneurysm formation was defined as a localized out-ward bulging of the vascular wall, whose diameter was 50% greater than the parent artery diameter. Aneurysm rupture was detected by performing daily neurological examinations, which was validated in a previous study [9]. To confirm aneurysm rupture, we perfused the mouse brain with bromophenol blue dye to visualize cerebral arteries. Rupture rate was defined as the number of mice with ruptured aneurysms divided by the total number of mice with any aneurysms [9]. Detailed methods of the aneurysm model and neurological symptom scoring are described in the Online Supplements.

We previously found that aneurysmal rupture occurred approximately starting from day 6–7 after aneurysm induction [9]. Thus, administration of MSC-derived MVs was started on day 6, which allowed us to detect the effects of MVs on aneurysm rupture rate without affecting the overall incidence of aneurysm formation. Thirty μL of MVs or vehicle (PBS) were intravenously administered through the jugular vein on day 6 and day 9 after aneurysm induction. To understand the involvement of E prostanoid 4 (EP4) receptor on the effect of MVs on aneurysmal rupture in vivo, 1 mg/kg of GW 627368X (Cayman Chemicals, Ann Arbor, MI), a selective EP4 antagonist, was administered intravenously simultaneously with MVs. In separate experiments, systemic distribution of PKH26-labled MVs was examined in mice 24 h after the initial MV administration, as described in the Online Supplements.

Toluidine Blue Staining and Quantification of Mast Cells

For quantification of total and activated mast cells accumulated in the brain, additional mice in all groups (N = 6–8) underwent aneurysm induction and were euthanized 2 days following MV administration. Mouse brains were cryostat cut into 8 μm-thickness slices and stained with 0.1% acid toluidine blue (Sigma-Aldrich). One cross-sectional slice, which included the middle cerebral artery, was used for mast cell quantification for each mouse. The activities of mast cells were determined by their morphology [16].

Mast Cell Activation and Treatment with MSCs or MVs

Cultures of mast cell were activated by incubation with calcium (Ca2+) ionophore A23187 (Sigma-Aldrich), and mast cell activation was assessed by measuring the release of tumor necrosis factor α (TNFα), a pro-inflammatory cytokine significantly involved in the pathogenesis of intracranial aneurysm [17, 18]. BMMCs or LAD2, seeded at 0.25 × 106 cells/well in 24-well plates, were incubated with 500 nmol/L (for BMMCs) or 250 nmol/L (for LAD2) of A23187 in serum-free culture media. After 15 h, cell supernatants were collected for TNFα measurements using mouse or human TNFα Quantikine ELISA kit (R&D Systems, Minneapolis, MN). BMMCs from two wells were pooled as one sample for total RNA extraction and quantitative real-time PCR. Dimethyl sulfoxide (DMSO, Sigma-Aldrich), 0.01–0.02% (v/v), was used as the control vehicle.

To determine the effects of MVs or MSCs on mast cell activation, mast cells were treated with 30 μL of MVs or co-cultured with MSCs in the presence of A23187. In the co-culture experiments to prevent cell-cell contact, we utilized a transwell system where 0.25 × 106 cells/well of MSCs were seeded and cultured overnight in the upper chamber. On the following day, the same number of mast cells was added in the bottom chamber with the addition of A23187. To study the role of PGE2 and EP receptors in the effect of MVs on mast cells, we utilized inhibitors, receptor antagonist, or exogenous PGE2 at different concentrations in vitro: NS-398 at 1, 10 μmol/L (Cyclooxygenase-2 (COX2) inhibitor, Cayman Chemicals), GW 627368X at 0.1, 1, 10 μmol/L (selective EP4 receptor antagonists, Cayman Chemicals) and PGE2 at 0.1, 1 μmol/L (Sigma-Aldrich).

Assays for Tryptase and Chymase Activity in BMMCs

We assessed tryptase activity using mast cell degranulation assay kit (IMM001, Millipore, Billerica, MA), according to the manufacturer’s recommendations. The cell lysates prepared for tryptase assay were also used for chymase activity assessment, which utilized a substrate, N-succinyl-ala-ala-pro-phe-pNA (AAPF-pNA, Sigma-Aldrich) as described previously with minor modification [19].

Statistics

The aneurysm rupture rate and the incidence of aneurysm formation were statistically analyzed by Fisher exact test. The symptom-free rate analysis was performed using the log-rank test. All other statistical comparisons were performed with student’s t-test or one-way ANOVA with Bonferroni correction, using GraphPad Prism version 5.0 (San Diego, CA) for Macintosh. Data were expressed as the mean ± SD. Statistical significance value was set at p < 0.05.

RESULTS

Quantification of Protein and Total RNA Contained in MVs and Internalization of MVs by BMMCs

Similar to previous studies [5, 8], MVs were visualized as multiple, approximately 200 nm, spheroid structures released from the surface of human MSCs under transmission electron microscopy (Figure 1A). Protein and total RNA contents in 30 μL of MVs, which was the therapeutic dose chosen in this study, were quantified as 27 ± 8 μg and 70 ± 24 ng respectively (Figure 1B), consistent with the results in previous studies [8].

Figure 1.

Figure 1

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Biological evaluation of human MSC-derived MVs. (A) Representative photographs of transmission electron microscopy of MVs. Upon serum deprivation, human MSCs release MVs (see arrows) as small circular membrane fragments from the cell surface. Scale bar = 2 μm. The insert shows the ultrastructure of purified MVs. Scale bar = 500 nm. (B) The quantification of proteins and total RNA contained in 30 μL of MVs, the therapeutic dose used in vivo and in vitro. (C–D) The internalization of PKH26-labeled MVs by mouse BMMCs after 15 h of incubation in the presence or absence of Ca2+ ionophore, A23187. (C) Representative photographs of confocal microscopy showing activated BMMCs incubated with PKH26-PBS (left) for non-specific binding, PKH26-labeled MVs (middle) and inactivated BMMCs with PKH26-labeled MVs (Right). Original magnification: ×400. Scale bar = 20 μm. (D) FACS analyses of internalization by activated BMMCs of PKH26-labeled MVs (red curve), PKH26- PBS (solid blue curve in panel a) or PKH26-labeled MVs pretreated with 1 μg/mL of anti-CD44 blocking antibody (dot curve in panel b) or IgG antibody (dot curve in panel c). Panel d shows the internalization of PKH26-labeled MVs (light blue dot curve) by inactivated BMMCs. In each panel, the gray area indicates the total cell number (DAPI positive). The bar graph shows the percentage of PKH26-positive BMMCs in each group on FACS analyses. Values are mean ± SD, n = 3. *p is significant versus PKH26- PBS and #p is significant versus inactivated BMMCs treated with PKH26-labeled MVs by ANOVA with Bonferroni correction.

Confocal microscopy demonstrated that PKH26-labeled MVs were internalized by BMMCs (Figure 1C). Cytofluorimetric analyses (FACS) showed that the internalization of PKH26-labeled MVs in activated BMMCs was two-fold higher than that in inactivated BMMCs (45 ± 4% versus 17 ± 1%, p = 0.0015, n = 3; Figure 1D), suggesting that the uptake of MVs was enhanced by mast cell activation. Cells treated with PKH26-PBS, used to detect non-specific staining, showed minimal uptake (0.2 ± 0.02%, n=3; Figure 1D). We previously reported that CD44 was expressed in human MSC-derived MVs and was critical for the incorporation of MVs in human alveolar epithelial type 2 cells [20]. However, in this study, MVs pre-treated with anti-CD44 blocking antibody did not significantly inhibit their incorporation in BMMCs, suggesting that other receptors may be responsible for MVs uptake by BMMCs (Figure 1D).

MSC-Derived MVs Stabilize Intracranial Aneurysms In Vivo

One dose of MVs (30 μL) or vehicle (PBS) was intravenously administered in mice at day 6 and day 9 after aneurysm induction, as shown in the diagram of the experimental protocol (Figure 2A). MVs treatment did not affect the overall incidence of aneurysm formation (Figure 2B), whereas it significantly reduced the rupture rate as compared to vehicle (42% versus 81%, p = 0.0095, n = 26; Figure 2C). The onset of aneurysm rupture was determined by daily neurological examination. As shown in Figure 2D, the difference in symptom-free rate between MVs and vehicle groups appeared from day 7 and gradually increased up to day 21 (p = 0.0044, n = 26). Mice that did not develop aneurysms were excluded from the rupture rate and symptom-free curve analyses. Figure 2E shows representative pictures of normal cerebral arteries (a), an unruptured aneurysm in an asymptomatic mouse with MVs treatment (b), and a ruptured aneurysm in a symptomatic mouse treated with vehicle (c). In summary, MVs intravenously administered after aneurysm formation prevented the rupture of intracranial aneurysm in mice.

Figure 2.

Figure 2

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Effects of intravenous administration of human MSC-derived MVs in the rupture of intracranial aneurysms. (A) Schematic diagram of experimental protocol to examine MVs treatment in a mouse model of aneurysm. (B–D) Incidence of aneurysm formation (unruptured and ruptured) (B), aneurysmal rupture rate (C) and symptom-free curve (Kaplan-Meier analysis curve) (D) were examined within 21 days after aneurysm induction in mice treated with vehicle (PBS) or MVs. In (D), the vertical dash lines indicate the time point of MVs or vehicle administration. Mice with aneurysm-free were excluded from graphs in (C) and (D). (E) Representative pictures of normal brain, unruptured and ruptured aneurysms with the whole view and magnified view at aneurysm site. *p < 0.01 by Fisher exact test; **p < 0.005 by log-rank test.

Additional animal experiments were performed to detect the distribution of PKH26-labeled MVs administered in mice on day 6 following aneurysm induction. As reported in previous studies, MVs may adopt the homing pattern of MSCs because MVs acquire the same repertoire of surface receptors and extracellular matrix binding proteins as the parent cells. We observed that MSC-derived MVs accumulated in lungs, liver and spleen (Supplemental Figure 1B–D) [21]. More importantly, MVs also accumulated in brain tissue cells in and adjacent to the aneurysmal artery (Supplemental Figure 1A), suggesting that MVs were able to pass through the blood brain barrier. The finding of MVs distribution in vivo suggested that MSC-derived MVs might traffic to the inflammatory site as well as have systemic effects.

Histological Assessment of the Immunomodulatory Effect of MVs on Mast Cells In Vivo

To investigate the effects of MVs on mast cells, we assessed the infiltration and activation of mast cells in the aneurysms and adjacent tissues in mice after the induction of intracranial aneurysm. Figure 3A illustrates the mast cells stained with toluidine blue in the vehicle and MVs treatment groups. Activated mast cells were defined by their morphology (>10% of the granules exhibiting fusion or discharge). Mast cells were rarely seen in the control mice without the presence of aneurysm. MVs administration reduced the number of total mast cells (p < 0.0001) as well as the number of activated mast cells (p < 0.0001) surrounding the aneurysmal cerebral arteries as compared to vehicle treatment (Figure 3C–F). These results suggest that the suppression of MVs on mast cell infiltration and activation at intracranial aneurysms may be one of the mechanisms underlying the stabilization of intracranial aneurysm by MVs.

Figure 3.

Figure 3

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Histological assessment of mast cell infiltration and activation at peri-vascular zone of intracranial aneurysms. The mast cells were counted as cells with metachromatic staining using toluidine blue in one cross-sectional slice including the middle cerebral artery, which is immediately distal of the bifurcation from the internal carotid artery, from each mouse. The activity of mast cells was determined by their morphology. (A) Representative micrographs of an activated mast cell (left) in the brain section of vehicle group and an inactivated mast cell (right) in the brain section of MVs treatment group. Scale bar = 50 μm. The number of total mast cells (B) and the number of activated mast cells (C) were counted on brain slices (one slice for each mouse) used for mast cell assessment or calculated as the number per 1 mm2. N = 6. *p indicates significance by ANOVA with Bonferroni correction.

MSC-Derived MVs Suppress A23187-Induced Mast Cell Activation

To identify the effects of MVs on mast cell activation, we performed experiments in vitro using primary culture of murine BMMCs and a human mast cell line, LAD2. Activation of mast cells induced by Calcium ionophore A23187 at an optimal concentration of 500 nmol/L for BMMCs or 250 nmol/L for LAD2 was determined by the quantification of TNFα release in culture media after 15 h incubation. Activated BMMCs or LAD2 showed a substantial increase in TNFα release (1247 ± 43 pg/mL for BMMCs; 2739 ± 1 pg/mL for LAD2) over control cells receiving the same volume of vehicle solution, DMSO. The exposure to A23187 for 15 h was not cytotoxic to mast cells (BMMCs and LAD2) as shown by > 95% viability with trypan blue staining (data not shown). Thirty μL of MSC-derived MVs administered to 0.25 ×106 of BMMCs or LAD2 cells with A23187-induced activation significantly decreased TNFα release by 77% and 55% respectively (Figure 4A and 4B). A double dose of MSC-derived MVs (60 μL) did not further decrease TNFα release from BMMCs or LAD2 (data not shown). Interestingly, the decrease in TNFα release was similar to treatment with MSCs co-cultured with mast cells (BMMCs or LAD2) at a ratio of 1:1 in a transwell plate, indicating that MVs derived from MSCs was as efficacious as MSCs on the suppression of mast cell activation. In addition, inactivated BMMCs did not release TNFα after MVs treatment. MVs isolated from normal adult human fibroblasts (NHF) were tested as a control to determine if the observed effect was specific to MSC-derived MVs. No significant reduction of TNFα release was observed in BMMCs or LAD2 treated with NHF-derived MVs (Supplemental Figure 2). Since LAD2 is a human cell line, we chose to only use mouse BMMCs for all subsequent experiments. Figure 4C demonstrated that either MSCs or MVs treatment markedly down-regulated the mRNA expression of TNFα in activated BMMCs compared to BMMCs treated with stimulant alone (p < 0.0001, Figure 4C), suggesting that the suppression of MVs or MSCs on the TNFα release was associated with a concomitant decrease in TNFα synthesis in the activated BMMCs.

Figure 4.

Figure 4

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Effects of MSC-derived MVs or MSCs on mast cell activation in vitro. BMMCs or LAD2 (250,000 cells/well cultured in 24-well plate), activated by incubating with A23187 (indicated as “Ca +”) for 15 h, were co-cultured with MSCs (250,000 cells/well seeded in transwells) or treated with MVs 30 μL. DMSO was used as vehicle. (A, B) The TNFα concentration in culture medium from BMMCs (A) and LAD2 (B). (C) The level of TNFα mRNA in BMMCs quantified by qRT-PCR analyses. BMMCs from two wells were pooled as one sample for assessing the activity of tryptase (D) and chymase (F) and the mRNA expression of MCP6 for tryptase (E), MCP4 (G) and MCP5 (H) for chymase. Values are mean ± SD, n = 3 – 6. Non-detectable value indicated as “ND”. #p < 0.05 by t-test analysis; *p is significant by ANOVA with Bonferroni correction.

Tryptase and chymase, the main serine proteinases expressed by mast cell, are important mediators in the development of aneurysm formation and rupture [3, 22]. In Figure 4D–H, administration of either MVs or MSCs decreased the activity of tryptase and chymase in activated BMMCs to basal levels (as shown in the controls). Although not statistically significant, both treatments numerically down-regulated the mRNA expression of tryptase (MCP6) and chymase (MCP4 and MCP5) in the activated BMMCs.

Increasing PGE2 Secretion Contributed to the Suppression of MVs on Mast Cell Activation

Since PGE2 secretion from MSCs has been previously found to be involved in the suppression of mast cell function in allergic diseases [18, 23], we investigated if MVs also mediated the suppressive effects by regulating PGE2 synthesis and its response in BMMCs. As shown in Figure 5A, PGE2 release by BMMCs was mildly increased after A23187-induced activation but it was significantly elevated following MVs treatment in activated BMMCs (53 ± 13 vs. 213 ± 6 pg/mL, p < 0.0001, n = 6). Figure 5B showed MVs treatment also up-regulated the mRNA expression of COX2, one of the key enzyme for PGE2 synthesis compared to cells without MVs treatment (p = 0.035). Co-culture of MSCs and BMMCs led to an increase of PGE2 level and COX2 mRNA in BMMCs to a greater extent than compared to MVs treatment on BMMCs. Administration of NHF-derived MVs as a control to activated BMMCs had no effect on PGE2 release (data not shown). The addition of a specific COX2 inhibitor (NS-398 at 1 μmol/L or 10 μmol/L) to BMMCs significantly inhibited the increase of PGE2 induced by MVs treatment (Figure 5C) and also eliminated the suppressive effect of MVs on TNFα release by BMMCs (Figure 5D). The results demonstrated that MVs mediated the up-regulation of PGE2 synthesis, which contributed to the inhibitory effects on mast cells activation.

Figure 5.

Figure 5

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PGE2 involved in the mechanism of MVs suppression on mast cells. (A, B) PGE2 concentration in culture media (A) and murine COX2 mRNA level (B) of BMMCs activated with A23187 (indicated as “Ca +”) in the absence or presence of MVs or MSCs. Cells incubated with DMSO alone was indicated as vehicle. (C, D) The addition of a specific COX2 inhibitor (NS-398 at 1 μmol/L or 10 μmol/L) eliminated the increase in PGE2 level (C) and the immunosuppressive effect of MVs on TNFα release (D) of activated BMMCs treated with MVs. Values are mean ± SD. Non-detectable value indicated as “ND”. N = 6. *p is significant by ANOVA with Bonferroni correction. #p < 0.05 by t-test analysis.

Altered mRNA Expression of E-Prostanoid Receptor 4 (EP4) in BMMCs Treated with MVs

To further identify the role of PGE2 in facilitating the suppression of MVs on mast cell activation, we examined if MVs were able to alter the expression profile of four EP receptors responsible for the regulation of PGE2 responsiveness in mast cells. BMMCs can express all four subtypes of PGE2 receptors, EP1-4. The primers we used to detect EPs expression were exclusively for murine. Thus, any EPs mRNAs potentially transferred by MVs from human MSCs were excluded in this evaluation. Activation of BMMCs dramatically increased the expression of EP2-4 and decreased the expression of EP1, as compared to inactivated BMMCs. MVs treatment caused further increase in the EP4 receptor mRNA expression (p = 0.043, n = 6); there was no significant changes detected in the mRNA for EP1-3 receptors (Figure 6A). Previous studies indicated that PGE2 exerts its anti-inflammatory effects through binding to EP4 on mast cells [24–26]. In the current study, the results suggest that MVs may enhance their PGE2-mediated suppressive effect on mast cell activation via up-regulating the expression of EP4 receptor. To further confirm the important role of EP4 receptor in the mechanism, we applied selective EP4 antagonist, GW 627368X, in our in vitro and in vivo experiments with MVs treatment. Figure 6B showed in vitro GW 627368X administration at 1 μmol/L or 10 μmol/L inhibited the suppression of TNFα release by BMMCs with MVs treatment. Similarly, the IV administration of GW 627368X (1 mg/kg) eliminated the protection by MVs against the rupture of intracranial aneurysm in vivo, showing no differences from vehicle group in rupture and symptom-free rate (Figure 6C). Our results revealed that the inhibitory effect of MVs was facilitated through the up-regulation and activation of EP4 receptors on mast cells.

Figure 6.

Figure 6

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The anti-inflammatory role of EP4 in mediating the immunosuppressive effect of MVs. (A) MVs treatment altered the mRNA expression of EP 1-4 receptors in activated BMMCs (stimulated with A23187 (indicated as “Ca +”). EP1 and EP3 mediate the pro-inflammatory effects of PGE2 on mast cells but EP4 mediates anti-inflammatory responses. N = 6. (B, C) The administration of a selective EP4 receptor antagonist (GW 627368X, indicated as “GWX”) eliminated the suppressive effects of MVs on TNFα release in activated BMMCs (B) as well as the protective effects of MVs against rupture of intracranial aneurysms in mice (C) (N = 8 in each group). #p < 0.05 by t-test analysis; **p is significant by ANOVA with Bonferroni correction; *p < 0.05 by Fisher exact test.

Role of Exogenous PGE2 in BMMCs Immunosuppression

We further investigated the immunosuppressive effect of PGE2 on mast cells using exogenous PGE2 at 0.1 μmol/L or 1 μmol/L administered to BMMCs in the presence of A23187. After 15 h of incubation, exogenous PGE2 reduced TNFα release (p < 0.0001; Figure 7A) and TNFα mRNA level (Figure 7B). In contrast to the effect of MVs treatment on EP4 up-regulation, the expression of all four EP receptor was suppressed by the treatment with 1 μmol/L of PGE2 (Figure 7C). These data indicate that elevated PGE2 levels mediated the immunosuppression on mast cell activation but did not directly result in the up-regulation of EP4 receptor expression found with MVs treatment.

Figure 7.

Figure 7

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Effects of exogenous PGE2 on mast cell activation and EPs receptor expression. (A) The levels of TNFα in culture medium from BMMCs stimulated with vehicle (DMSO) or A23187 (indicated as “Ca +”; 500 nmol/L), in the absence or presence of PGE2 at 0.1 μmol/L or 1 μmol/L. In the parallel experiment with (A), cells treated with 1 μmol/L of PGE2 were collected for the quantitative measurement of TNFα mRNA (B) and EP 1-4 receptor mRNA (C). N = 6. Values for EP2 mRNA are divided by 1000. ***p is significant by ANOVA with Bonferroni correction.

DISCUSSION

The main findings of this study are summarized as follows: 1) In a mouse model of intracranial aneurysm, intravenous administration of human MSC-derived MVs reduced aneurysmal rupture rate by 39% (Figure 2), which was associated with reduced infiltration and activation of mast cells around the aneurysm arteries (Figure 3); Trafficking experiments demonstrated the presence of MVs around the aneurysm site 24 h following administration (Supplemental Figure 1); 2) Similar to MSCs, MVs suppressed mast cell activation in vitro by significantly decreasing TNFα secretion in both BMMCs and LAD2 cells (Figure 4A–B) as well as the activities of tryptase and chymase in BMMCs (Figure 4D and 4F); 3) The suppressive effect on mast cells was mediated through a PGE2-dependent mechanism. MVs treatment induced a higher level of PGE2 production in BMMCs (Figure 5A) and, perhaps more importantly, also enhanced EP4 receptor expression, which may be associated to the anti-inflammatory responses in BMMCs (Figure 6A). MVs-induced inhibition on TNFα production in BMMCs was abolished by the addition of a COX2 inhibitor (Figure 5D) or a selective EP4 antagonist, GW 627368X (Figure 6B); 4) And, administration of a selective EP4 antagonist with MVs in mice eliminated the therapeutic effect of MVs in aneurysm rupture in vivo (Figure 6C).

In previous studies, MSCs have been used as a therapeutic to prevent the formation and rupture of aneurysms due to their capacity in promoting vascular regeneration and repair, decreasing inflammatory cytokine production, suppressing the activities of matrix metalloproteinase (MMP), and preserving the elastin content in arterial walls [27, 28]. Recently MVs released from MSC were found to have similar functional effects as their parent cells through the active transfer of a specific group of surface receptors, proteins, mRNA and bioactive lipids [29]. An increasing number of studies have shown that MSC-derived MVs have similar capacity as MSCs for immunomodulation of macrophages and regulatory T cells as well as a cytoprotective effect on ischemia/reperfusion injury in animal models [5, 30]. In our study, we demonstrated that human MSC-derived MVs significantly reduced the rupture rate of intracranial aneurysm in mice, which was associated with fewer mast cells infiltration into the aneurysmal wall. In addition, trafficking studies with PKH26-labeled MVs showed that in vivo MVs accumulated in the lungs, liver and spleen, as well as, more importantly, in brain tissue cells adjacent to the aneurysmal arteries, e.g., middle cerebral artery, as shown in the Supplemental Figure 1. To our knowledge, the application of MVs derived from MSCs has never been investigated using in vivo aneurysm models. Our study is the first one to report the therapeutic effects of MVs on aneurysmal rupture as well as the first one to present the modulation of MVs on mast cell activity in the progression of aneurysmal rupture.

Inflammation is involved in the pathogenesis of intracranial aneurysm. Inflammatory cells, such as macrophages, T lymphocytes and natural killer cells were all found to have a detrimental effect on the aneurysmal arteries [31]. Recently, the important role of mast cells in the pathogenesis of aneurysms, especially in the progression to aneurysmal rupture, has been established by previous studies using human tissues or animal models. Our group previously reported that mast cell expression in human tissues of intracranial aneurysm was markedly upregulated in ruptured aneurysms as compared to unruptured aneurysms [1]. Ishibashi et al. applied inhibitors of mast cell degranulation in a rat model of intracranial aneurysm to effectively attenuate vascular destruction in aneurysmal walls [2]. In the experimental models of abdominal aortic aneurysm (AAA), genetic deficiency of mast cells, pharmacological inhibition of mast cell degranulation, or absence of mast cell-specific proteases all effectively attenuated or abolished AAA growth [4, 32]. Therefore, mast cells can be seen as an ideal cell target for the treatment of aneurysms. Our study explored the potential relations of MVs with mast cell activation in the context of intracranial aneurysmal rupture. The histological assessment of total and activated mast cells in mouse brains revealed that MVs treatment suppressed the infiltration and activation of mast cells around aneurysmal arteries. The suppressive effect of MVs on mast cell activity in brain may potentially play a significant role in the prevention of aneurysmal rupture.

In support of our results of the effect of MVs on mast cells in vivo, we found that MVs treatment suppressed mast cell activation in vitro by decreasing the level of cytokine release and protease activity. TNFα, among the main pro-inflammatory cytokines released by mast cells, is associated with the development of intracranial aneurysm by promoting the degradation of extracellular matrix and apoptosis of smooth muscle cells in the vascular wall [17]. Previous studies, conducted in co-culture experiments of MSCs with mast cells in transwell plates, showed a significant decrease of TNFα level despite a lack of cell-to-cell contact [18, 23]. In our in vitro study, we found a reduction of TNFα release from mast cells after MVs treatment, similar to MSCs (Figure 4A–B). It suggests that MVs might act as one of the mediators for MSCs to interact with mast cells. We also demonstrated that MVs treatment effectively decreased the activity of chymase and tryptase, two mast cell-specific proteases, in activated mast cells. These two proteases are essential in the pathogenesis of aneurysms [3] because they are involved in the proteolysis of a wide range of factors (e.g., matrix proteins and growth factors), and the activation of various proteinases such as MMPs, potentially resulting in the degeneration of the extracellular matrix in arterial walls [22, 33]. The inhibition on protease activities may be another potential mechanism underlying the therapeutic effects of MVs on intracranial aneurysm rupture.

Interestingly, our study demonstrated that MVs suppressed mast cell activation in part through a COX2-PGE2-dependent mechanism. PGE2 exert anti-inflammatory actions by selectively suppressing Th1 differentiation, B-cell functions, T-cell activation and directly suppressing the production of multiple pro-inflammatory cytokines [11]. We detected an increased level of PGE2 release and the up-regulation of COX2 mRNA in activated mast cells following MVs treatment. However, other studies have reported that PGE2 can exert both pro- and anti-inflammatory effects on various immune cells [11, 12]. To find out if increased PGE2 and COX2 expression were associated with MVs immunosuppressive effect on mast cells, we deactivated COX2 by using NS-398, which markedly reduced PGE2 release and eliminated the suppressive effect of MVs on mast cell activation, suggesting that increased COX2 expression by MVs contributed to the immunosuppression on mast cells. The effect of MVs on other enzymes involved in PGE2 synthesis and degradation (e.g., microsomal prostaglandin E synthases and 15-hydrooxyprostaglandin dehydrogenase) is an area of future studies.

EP receptors are another factor influencing the function of PGE2 on target cells. For instance, hemodynamic stress induces intracranial aneurysms via the activation of PGE2-EP2 signaling in the endothelium [34]. The heterogeneous effects of PGE2 are partially determined by the expression pattern of four EP receptor subtypes (EP1-4) [11]. Mast cells are able to express four subtypes (EP1-4) [24, 35]. Therefore, we studied the four EPs (EP1-4) expressed on mast cells treated with MVs. Our RT-PCR analysis revealed that MVs significantly up-regulated the EP4 gene expression in activated mast cells, whereas EP3 expression was slightly decreased but not significantly. Using the selective EP4 antagonist, GW 627368X, we found that EP4 was critical for MVs suppression of mast cell activation in vitro and also for MVs protection against aneurysmal rupture in vivo. Similarly, Brown et al. identified MSCs-derived PGE2 mediated the suppression of mast cells by binding EP4 receptor while the presence of EP1, EP2 or EP3 receptor was not critical [18]. In a sepsis model, Nemeth et al. found that MSCs re-programmed macrophage to an anti-inflammatory M2 phenotype by releasing PGE2, which acted on the EP2 and EP4 receptors on macrophages [36]. In contrast to EP4 receptor, EP3 receptor is responsible for the activation of bone marrow-derived mast cells, and this mechanism underlies PGE2-induced vascular permeability [37]. The differential expression of the PGE2 receptors allows immune cells to adapt to the environment. Our study was the first to report that MSC-derived MVs upregulated EP4 expression, which facilitates their suppressive effects on activated mast cells.

To further identify if the increasing level of PGE2 induced by MVs treatment was responsible for the EP receptor expression profile, we administered exogenous PGE2 and examined the effect on cytokine release and EP receptor expression in activated mast cells. The results demonstrated that exogenous PGE2 had a suppressive effect on TNFα release and mRNA expression in mast cells, consistent with the results in BMMCs with MVs or MSCs treatment. Interestingly, we did not observe similar changes in EP receptor expression induced with exogenous PGE2 as we detected with MVs treatment on mast cells. Therefore, it suggests that MVs regulate the expression of EP receptors on activated mast cells via a different mechanism from PGE2-induced regulation on EP receptors.

There are limitations to the current study. First, we did not perform a dose response of MVs in terms of aneurysmal rupture in the intracranial aneurysm model or explore the role of MVs on aneurysm formation. It is conceivable that there may be an effect of MVs administration on aneurysm formation as well. Second, it is unclear the long-term effect of MVs on aneurysmal rupture rate in vivo. Further studies with longer periods of follow-up may enhance the clinical significance of MVs treatment on aneurysms. Finally, other possible biological mechanisms involved in the anti-inflammatory properties of MVs need further work.

CONCLUSION

In conclusion, administration of MSCs-derived MVs prevented the rupture of intracranial aneurysm, in part by suppressing mast cell activation via increasing the release and synthesis of PGE2 as well as up-regulating the expression of EP4 receptor on mast cells. More importantly, MSC-derived MVs were as effective as MSCs as a therapeutic, suggesting a possible alternative to using cells given the potential limitations of any stem cell-based therapy.

Supplementary Material

Figures
TEXT/Table

Acknowledgments

We thank Drs. Arnold Kirshenbaum and Dean Metcalfe for kindly providing the human mast cell line LAD2. The generation of the LAD2 cell line was funded by a grant from National Institute of Allergy and Infection Diseases. We also thank Drs. Mao Mao, Yinghong Xiao, Yinggang Zhu and Antoine Monsel for their technical supports.

Brief acknowledgment of grants: R01NS055876 and R01NS082280 from NIH/NINDS (Dr. Tomoki Hashimoto). R01HL-113022 from NHLBI (Dr. Jae-Woo Lee).

Footnotes

Author contributions:

Jia Liu: Conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing.

Atsushi Kuwabara: Conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing.

Yoshinobu Kamio: Collection of data.

Shuling Hu: Collection of data.

Jeonghyun Park: Collection of data.

Tomoki Hashimoto: Conception and design, financial support, data analysis and interpretation, and manuscript editing.

Jae-Woo Lee: Conception and design, financial support, data analysis and interpretation, manuscript writing and final approval.

Disclosures of Potential Conflicts of Interest

None.

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