Alix-mediated selective packaging of β-catenin into extracellular vesicles enhances their proangiogenic function

TL;DR

Alix helps pack β-catenin into extracellular vesicles, which boosts their ability to promote blood vessel growth.

Contribution

Alix selectively packages β-catenin into EVs, enhancing their proangiogenic function.

Findings

Alix-overexpressing MSC-derived EVs promote angiogenesis in vitro and in vivo.

Alix-knockdown EVs suppress angiogenesis.

Alix enhances β-catenin enrichment in EVs.

Abstract

The function of extracellular vesicles (EVs) is determined by the molecular cargo they carry from their parent cells. Although apoptosis-linked gene 2–interacting protein X (Alix) is known to regulate EV cargo loading and functional properties, the specific mechanisms underlying its role in mediating β-catenin sorting and function remain unclear. In this study, we first observed the colocalization of Alix and β-catenin through immunofluorescence staining. To assess whether the interaction between Alix and β-catenin affects the function of mesenchymal stem cell (MSC)–derived EVs, we generated Alix-knockdown and Alix-overexpressing MSCs via viral transduction. Analysis of secreted EVs revealed that those derived from Alix-overexpressing MSCs promoted angiogenesis both in vitro and in a mouse model of hindlimb ischemia, whereas EVs from Alix-knockdown MSCs suppressed angiogenesis. Mechanistically, we confirmed that the Alix–β-catenin interaction selectively enhances β-catenin enrichment within EVs. In conclusion, our findings demonstrate that Alix plays a critical role in selectively packaging β-catenin into EVs, thereby enhancing their proangiogenic potency.

Figures

Full text

Mesenchymal stem cells (MSCs) possess self-renewal capabilities and the potential for multidirectional differentiation. They are widely distributed throughout the human body and are present in tissues, such as the bone marrow, adipose tissue, placenta, and umbilical cord (1). In addition, extracellular vesicles (EVs) secreted by MSCs are considered promising sources of natural therapeutic agents. Exosomes, a subtype of EVs, can deliver a variety of biomolecules, facilitate intercellular communication, and perform numerous biological functions (2, 3). Owing to the protective nature of their biological membranes, EVs are less susceptible to enzymatic degradation and other damaging substances, making them more effective than direct delivery (4). As cellular components change, the composition of EVs may also shift, potentially altering their function. Therefore, understanding the mechanisms of component selection and packaging into EVs is essential.

Exosomes are released from cells following the fusion of multivesicular bodies with the plasma membrane (5). The endosomal sorting complex required for transport (ESCRT) is essential for EV biogenesis (6, 7). Alix (apoptosis-linked gene 2–interacting protein X), also known as programmed cell death 6–interacting protein, is an auxiliary protein of the ESCRT-III complex and plays a crucial role in EV biogenesis (8, 9). For example, compared with control cells, human embryonic kidney 293T (HEK293T) cells lacking Alix showed no significant difference in EV release (10). Although Alix does not affect the quantity of EVs produced, it regulates the packaging of cargo into EVs, thus affecting the material secreted by the cell through EVs. The overexpression of Alix in induced pluripotent stem cell (iPSC)–derived exosomes increased both protein content and protective function, promoting angiogenesis and enhancing disease treatment and tissue repair (11). Knockdown (KD) of Alix in MSCs derived from corneal stromal stem cells blocks miRNA packaging into EVs, inhibits the regenerative function of EVs, and reduces their ability to prevent scar formation (12). Therefore, Alix can modulate EV function by regulating the cargo composition, thus influencing the fate of both EV-secreting and recipient cells. This effect can be modulated by adjusting the Alix level in EV-secreting cells.

Alix-mediated exosomes derived from iPSCs have been shown to regulate angiogenesis (11). However, the effects of Alix-mediated EV secretion from MSCs on angiogenesis remain unclear. β-catenin interacts with CHMP4B, a component of the ESCRT complex, and CHMP4B KD reduces β-catenin incorporation into EVs (13). Alix also interacts with CHMP4B (14); however, whether Alix directly interacts with β-catenin remains unknown. β-catenin is a key component of the canonical Wnt signaling pathway, which is closely associated with angiogenesis (3, 15). Upon activation of the Wnt pathway, β-catenin translocates to the nucleus and activates the expression of angiogenesis-related genes, thereby promoting angiogenesis (16, 17, 18). Nevertheless, it remains uncertain whether the proangiogenic effects of Alix-regulated EVs are mediated through the Wnt/β-catenin signaling pathway.

In this study, we demonstrated that Alix interacts with β-catenin and plays a crucial role in mediating the sorting of β-catenin into EVs. Our study reveals a novel mechanism by which Alix regulates the angiogenic function of EVs derived from MSCs. These findings suggest that the targeted manipulation of MSCs can lead to the production of EVs with enhanced therapeutic potential for disease treatment and tissue repair.

Results

Characterization of Alix-KD human placenta–derived MSC EVs

Previous studies have shown that the auxiliary protein Vps4A, a component of the ESCRT-III complex, promotes the localization of β-catenin to the plasma membrane and its incorporation into EVs through interactions with both β-catenin and CHMP4B (13). In addition, an interaction between CHMP4B and Alix has been reported (14). On the basis of these findings, we first examined the colocalization of endogenous Alix and β-catenin in human placenta–derived MSCs (hP-MSCs) using an immunofluorescence (IF) assay (Fig. 1A). To further substantiate this observation, multiple independent fields of view were analyzed, and the degree of colocalization between Alix and β-catenin was quantified by calculating Pearson’s correlation coefficients (Fig. S1). The consistent colocalization observed across different fields supports a close spatial association between Alix and β-catenin in hP-MSCs.Figure 1Characterization of EVs derived from Alix-KD-hP-MSCs. A, confocal colocalization analysis was performed on hP-MSCs to visualize Alix (green) and β-catenin (red), with nuclei stained with DAPI. The scale bar represents 50 μm. B, RT‒qPCR analysis confirmed that Alix mRNA in hP-MSCs was decreased after Alix KD. C, Western blot analysis confirmed that Alix expression in hP-MSCs was decreased by Alix KD. The full-length blots are presented in Fig. S13. D, DLS was used to quantify the representative size distribution of the EVs. E, TEM revealed that the EVs were cup shaped. The scale bar represents 100 nm. Data are representative of three independent experiments (n = 3). Statistical significance in B was determined using Student’s t test; ∗∗∗∗p < 0.0001. Shaded areas and error bars represent the mean ± SD. DAPI, 4′-6-diamidino-2-phenylindole; DLS, dynamic light scattering; EV, extracellular vesicle; hp-MSC, human placenta–derived mesenchymal stem cell; KD, knockdown; MSC, mesenchymal stem cell; qPCR, quantitative PCR; TEM, transmission electron microscopy.

β-catenin is a key component of the canonical Wnt signaling pathway, and numerous studies have highlighted its role in angiogenesis [27–29]. However, whether the interaction between Alix and β-catenin influences the function of EVs derived from hP-MSCs remains unclear. To investigate this, we generated Alix-KD hP-MSCs via viral transduction. Control cells were transduced with a nonspecific control vector (Ctrl-KD-hP-MSCs). Alix-KD-hP-MSCs were confirmed by quantitative PCR (qPCR) and Western blot. Both the Alix mRNA level and protein expression were reduced in the Alix-KD-hP-MSCs (Figs. 1, B and C; Fig. S2). EVs regulated by Alix were then isolated from hP-MSCs by collecting the culture medium followed by ultracentrifugation.

The characteristics of the EVs were assessed via Western blot, dynamic light scattering (DLS), and transmission electron microscopy (TEM). EVs derived from Ctrl-KD-hP-MSCs and Alix-KD-hP-MSCs were named Ctrl-KD-EVs and Alix-KD-EVs, respectively. Western blot analysis confirmed the presence of the EV markers Alix and CD9 and the absence of the negative marker GM130 (Fig. 1C). Notably, Alix KD markedly reduced β-catenin levels in EVs derived from hP-MSCs (Figs. 1C, S2). Given the established role of MSC-derived EVs in angiogenesis, we next examined whether Alix-mediated regulation of MSC-derived EVs is associated with alterations in angiogenesis-related cargo. While Alix depletion did not affect fibroblast growth factor 2 (FGF2) levels in EVs, vascular endothelial growth factor A (VEGFA) levels were modestly increased in Alix-KD-EVs compared with control EVs (Fig. S3A). In contrast, qPCR analysis revealed significant reductions in the angiogenesis-related miRNAs, miR-21 and miR-126, in Alix-KD-EVs (Fig. S3B). DLS analysis revealed that the average diameter of the EVs was approximately 100 nm (Fig. 1D). Furthermore, TEM images revealed that the isolated EVs were round, membrane-bound vesicles (Fig. 1E). In addition, nanoparticle tracking analysis demonstrated that Alix-KD-EVs exhibited a modestly increased peak particle size compared with Ctrl-KD-EVs (Fig. S4A), whereas the total number of EVs released from Alix-KD hP-MSCs was not significantly different from that of control cells (Fig. S4B). Collectively, these results confirmed the successful isolation of Alix-KD hP-MSC–derived EVs and indicated that Alix KD selectively alters EV cargo composition without significantly affecting EV release.

Alix-KD EVs attenuated angiogenic capacity in vitro

In recent years, there has been increasing research on the regulation of EV biogenesis by Alix, particularly its role in regulating EV-mediated angiogenesis. Alix-regulated EVs derived from iPSCs have been shown to increase angiogenic potential (11). Similarly, EVs derived from MSCs with Alix knockout, which are derived from corneal stromal stem cells, also lose their regenerative ability (12). To investigate whether Alix influences the angiogenic potential of EVs derived from hP-MSCs, we conducted scratch wound healing and tube formation assays using human umbilical vein endothelial cells (HUVECs). Ctrl-EVs and Alix-KD-EVs labeled with the red fluorescent dye DiI were added to the HUVEC culture system and incubated for an additional 24 h. Microscopic imaging revealed that red fluorescence was localized in the cytoplasm (Fig. S5), indicating successful uptake of the EVs by the HUVECs. After incubation with hP-MSC-EVs for 24 h, the wound closure ratio in the Alix-KD-EV group was markedly lower than that in the Ctrl-EV group (Fig. 2, A and B). In the tube formation assay, HUVECs stimulated with Alix-KD-EVs formed fewer capillary-like structures on Matrigel (Fig. 2, C and D). To explore the underlying mechanisms, we examined the expression levels of proangiogenic genes in HUVECs. Compared with Ctrl-EVs, Alix-KD-EVs significantly reduced the mRNA expression of angiopoietin-1 (ANG-1), VEGFA, and vascular endothelial growth factor receptor 2 (VEGFR2) (Fig. 2E). Collectively, these results suggest that EVs derived from Alix-KD hP-MSCs lost their proangiogenic capacity in vitro.Figure 2EVs from Alix-knockdown hP-MSCs suppress the angiogenic capacity of HUVECs in vitro. A, scratch wound healing assay of HUVECs treated with 100 μg/ml corresponding EVs for 0 and 24 h. The scale bar represents 100 μm. B, migration ratio statistics for each group. C, representative images of the tube formation assay of HUVECs treated with 100 μg/ml corresponding EVs for 24 h. The bar represents 100 μm. D, quantification of the total number of branches and the total length of capillary-like tube structures in three random fields for each group. E, mRNA expression levels of ANG-1, VEGFA, and VEGFR2 in HUVECs treated with 100 μg/ml corresponding EVs for 24 h. Relative gene expression was normalized to that of GAPDH, and the data were analyzed via the 2^−ΔΔCt^ method. Bars represent the mean ± SD from three independent experiments. For B, D, E, statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple-comparison test. Statistical significance is indicated as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. EV, extracellular vesicle; hp-MSC, human placenta–derived mesenchymal stem cell; HUVEC, human umbilical vein endothelial cell; VEGFA, vascular endothelial growth factor A; ANG-, angiopoietin-1; VEGFR2, vascular endothelial growth factor receptor 2.

Alix-overexpressing EVs enhanced angiogenic capacity in vitro

These results suggest that Alix KD weakens the proangiogenic properties of EVs secreted by hP-MSCs. To further explore the role of Alix in modulating EV function, Alix-overexpressing hP-MSCs (Alix-OE-hP-MSCs) were generated via viral transduction. Control cells were transduced with a nonspecific control vector (Ctrl-OE-hP-MSCs). Alix-OE-hP-MSCs were confirmed via qPCR and Western blot. Both Alix mRNA levels and protein expression were elevated in Alix-OE-hP-MSCs (Fig 3, A and B; Fig. S6A). In contrast, the expression level of β-catenin was not significantly altered in Alix-OE-hP-MSCs relative to controls (Fig. S6A). EVs were isolated from the culture media of Ctrl-OE-hP-MSCs and Alix-OE-hP-MSCs through ultracentrifugation and designated Ctrl-OE-EVs and Alix-OE-EVs, respectively. Consistent with the aforementioned methods, Western blotting confirmed the presence of the EV markers, Alix and CD9, and the absence of the negative control protein, GM130 (Fig. 3B). Notably, Alix overexpression significantly increased β-catenin levels in EVs derived from hP-MSCs (Figs. 3B; Fig. S6B). As shown by the results of the DLS analysis, the diameter of the EVs was approximately 100 nm (Fig. 3C), and the TEM images revealed that the isolated EVs were round, membrane-bound vesicles (Fig. 3D). EVs derived from Alix-KD hP-MSCs inhibited cell migration, tube formation, and angiogenesis-related gene expression. To further evaluate the role of Alix in EV function, we conducted parallel experiments using Alix-OE-EVs. Ctrl-OE-EVs and Alix-OE-EVs labeled with the red fluorescent dye DiI were added to the HUVEC culture system and incubated for 24 h. Microscopic imaging revealed that red fluorescence localized within the cytoplasm (Fig. S7), confirming successful uptake of the EVs by the HUVECs. After 24 h of incubation, the wound closure ratio was significantly greater in the Alix-OE-EV group than in the Ctrl-OE-EV group (Fig. S8, A and B). In the tube formation assay, HUVECs treated with Alix-OE-EVs formed significantly more capillary-like structures on Matrigel (Fig. S8, C and D). In addition, compared with Ctrl-OE-EV treatment, Alix-OE-EV treatment upregulated the mRNA levels of the proangiogenic genes, ANG-1, VEGFA, and VEGFR2 (Fig. S8E). Collectively, these findings indicate that Alix modulates the angiogenic potential of EVs derived from hP-MSCs in vitro.Figure 3Characterization of EVs derived from Alix-OE-hP-MSCs. A, detection of Alix mRNA levels in hP-MSCs following Alix overexpression via RT‒qPCR. B, measurement of Alix expression in hP-MSCs and their derived EVs via Western blotting. The full-length blots are presented in Fig. S14. C, representative size distribution of EVs quantified by dynamic light scattering. D, transmission electron microscopy revealed that the EVs were cup shaped. The scale bar represents 100 nm. Data are representative of three independent experiments (n = 3). Statistical significance in B was determined using Student’s t test; ∗∗p < 0.01. Shaded areas and error bars represent the mean ± SD. Alix-OE-hP-MSCs, Alix-overexpressing human placenta–derived mesenchymal stem cell; EV, extracellular vesicle; qPCR, quantitative PCR.

Alix-OE EVs enhanced angiogenesis in vivo

To investigate the role of Alix in regulating the angiogenic capacity of hP-MSC–derived EVs in vivo, a hindlimb ischemia model was established in transgenic Vegfr2-luc mice. In this model, the firefly luciferase (Fluc) gene is expressed under the control of the Vegfr2 promoter, allowing bioluminescence intensity to serve as an indicator of angiogenesis (19). Using this approach, we monitored angiogenesis in different groups and observed Fluc signals in all groups, with the strongest signal detected in the Alix-OE-EV group (Fig. 4, A and B). In contrast, the signals in the Alix-KD-EV group were significantly weaker than those in the Ctrl-EV group (Fig. S9, A and B). Histological examination at 14 days revealed enhanced neovascularization in the ischemic tissues. CD31 immunostaining revealed a marked increase in microvascular density in the Alix-OE-EV group, whereas the density in the Alix-KD-EV group was significantly lower than that in the Ctrl-EV group (Fig 4, C and D; Fig. S9, C and D), which was consistent with the bioluminescence imaging (BLI) results. Together, these results suggest that therapy based on Alix-regulated hP-MSC–derived EVs influences endogenous angiogenesis in ischemic tissues.Figure 4EVs from Alix-OE-hP-MSCs promote angiogenesis in ischemic hindlimbs in vivo. A, in vivo angiogenesis status after treatment with Alix-OE-EVs or Ctrl-OE-EVs was monitored by tracking Vegfr2-luc expression via bioluminescence imaging (BLI) in a mouse hindlimb ischemia model. The signal activity was expressed as photons/second/cm^2^/steradian. B, the angiogenesis trend in ischemic hind limbs was quantitatively analyzed via Fluc signals. C, immunofluorescence staining for CD31 (red) was performed on day 14, with the nuclei counterstained with DAPI. The bar represents 100 μm. D, capillary density in the ischemic limbs of each group was quantified. The data represent the means ± SD (n = 5). Statistical analysis for B was performed using two-way ANOVA followed by Tukey’s multiple-comparison test, and statistical analysis for D was performed using one-way ANOVA followed by Tukey’s multiple-comparison test. Statistical significance is indicated as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ∗p < 0.05 versus PBS and ^&^p < 0.05 versus Ctrl-OE-EVs. Alix-OE-hP-MSC, Alix-overexpressing human placenta–derived mesenchymal stem cell; DAPI, 4′-6-diamidino-2-phenylindole; EV, extracellular vesicle.

To further evaluate the therapeutic potential of Alix-regulated EVs derived from hP-MSCs, we examined the morphological changes in muscle tissues via histological analysis. Hematoxylin and eosin (H&E) staining was performed on day 14 to assess injured hindlimb tissues via light microscopy. Compared with the Ctrl-EV group, the Alix-KD-EV group failed to exhibit improved tissue damage, whereas compared with the Ctrl-OE-EV group, the Alix-OE-EV group presented significantly fewer necrotic fibers and inflammatory cells in injured tissues (Fig. S10A). To assess fibrosis, Masson’s trichrome staining was conducted on day 28. Compared with the control group, the Alix-OE-EV group presented a marked reduction in the fibrotic area (Fig. S10, B and C). Consistent with the H&E staining results, Alix-KD-EVs did not alleviate fibrosis to the same extent as the control. Among the treatments, Alix-OE-EVs were the most effective at ameliorating hindlimb ischemia.

Alix mediates the interaction with β-catenin

To further explore the mechanism by which Alix-derived EVs derived from hP-MSCs influence angiogenesis, we confirmed the interaction between Alix and β-catenin by constructing HA-tagged Alix and FLAG-tagged β-catenin fusion proteins. Their interaction was validated through both IF and coimmunoprecipitation assays (Fig. 5, A and B). Given the reported association of CHMP4B with both Alix and β-catenin, we next assessed whether CHMP4B is required for the Alix–β-catenin interaction. To this end, CHMP4B was knocked down in HEK293T cells, followed by coimmunoprecipitation analysis using an anti-FLAG antibody. Compared with control cells, CHMP4B KD did not result in a significant change in the amount of Alix coprecipitated with β-catenin (Fig. S11). These findings indicate that the interaction between Alix and β-catenin occurs independently of CHMP4B. Alix is a marker of EVs, and determining whether its interaction with β-catenin influences the sorting of β-catenin into EVs is critical. Our previous results revealed a significant reduction in β-catenin levels in Alix-KD-EVs compared with those in Ctrl-EVs (Fig. 1C) and a corresponding increase in β-catenin levels in Alix-OE-EVs compared with those in Ctrl-OE-EVs (Fig. 3B). Collectively, these findings suggest that Alix promotes the sorting of β-catenin into EVs through its interaction with β-catenin.Figure 5Alix mediates the interaction with β-catenin. A, HeLa cells were transiently transfected with 3HA-Alix (green) and 3FLAG-β-catenin (red). After 48 h, the cells were fixed and stained with a rabbit anti-HA antibody, followed by incubation with a tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit secondary antibody and a mouse anti-FLAG antibody, followed by incubation with an isothiocyanate-conjugated goat anti-mouse secondary antibody. The bar represents 50 μm. B, HEK293T cells (4 × 10^6^) were transfected with 3HA-Alix and 3FLAG-β-catenin, and coimmunoprecipitation was performed with the FLAG antibody after 48 h. Cell lysates and immunoprecipitates were analyzed via Western blotting with anti-FLAG and anti-HA antibodies. The full-length blots are presented in Fig. S15. Alix, apoptosis-linked gene 2–interacting protein X; HEK293T, human embryonic kidney 293T cell line.

Alix-KD EVs attenuate angiogenesis by inhibiting the Wnt/β-catenin signaling pathway

Alix regulates the incorporation of β-catenin into EVs derived from hP-MSCs and modulates their proangiogenic activity. Previous studies have demonstrated that EVs from human umbilical cord MSCs promote the nuclear translocation of β-catenin, thereby activating Wnt/β-catenin signaling and facilitating wound healing (20). Based on these findings, we hypothesized that Wnt/β-catenin signaling may mediate the biological effects of Alix-regulated hP-MSC–derived EVs on angiogenesis. Consistent with this hypothesis, our data revealed that Alix-KD-EVs reduced the nuclear accumulation of β-catenin (Fig. 6, A and B). HUVECs were treated with equal amounts of Ctrl-EVs or Alix-KD-EVs for 24 h, and whole-cell lysates were collected. Western blot analysis revealed that Alix-KD-EVs inhibited Wnt/β-catenin signaling activation, leading to a rapid decrease in the expression of p-GSK3β and its downstream target gene VEGFA, whereas GSK3β levels remained unchanged (Fig. 6C). Furthermore, consistent with the reduced β-catenin content in Alix-KD-EVs compared with that in Ctrl-EVs (Fig. 1C), treatment with Alix-KD-EVs also lowered β-catenin levels in HUVECs. Similarly, treatment of HUVECs with Alix-KD-EVs at equivalent doses inhibited the activation of Wnt/β-catenin signaling, leading to a significant reduction in the mRNA expression of the downstream genes, cyclin D1 and c-Myc (Fig. 6D). In addition, compared with the control EV–treated group, incubation with Alix-KD-EVs led to a significant reduction in VEGFR2 expression in HUVECs, whereas no significant change was observed in VEGFR1 expression (Fig. S12). We further examined the activation status of the AKT and mitogen-activated protein kinase–extracellular signal–regulated kinase (ERK) signaling pathways by analyzing the levels of AKT1, p-AKT1, ERK, and p-ERK following EV treatment. No significant differences were detected in the p-AKT1/AKT1 or p-ERK/ERK ratios between the control EV and Alix-KD-EV treatment groups, indicating that these pathways were not detectably altered under the experimental conditions tested (Fig. S12). In summary, these results indicate that Alix-KD-EVs downregulate nuclear β-catenin protein levels, inhibit the Wnt signaling pathway, and suppress its transcriptional activity, thereby reducing the expression of angiogenesis-related genes.Figure 6Alix-KD-EVs attenuate angiogenesis by inhibiting Wnt/β-catenin signaling. A, HUVECs were treated with 100 μg/ml Ctrl-EVs or Alix-KD-EVs for 24 h. The nuclear translocation of β-catenin (red) was assessed through immunofluorescence staining. Nuclei were stained with DAPI. The scale bar represents 20 μm. B, cytoplasmic and nuclear fractions were prepared from HUVECs treated with 100 μg/ml Ctrl-EVs or Alix-KD-EVs for 24 h. β-catenin protein levels were measured via Western blotting. C, Western blotting was performed to quantify β-catenin, phosphorylated GSK3β (p-GSK3β), total GSK3β, and VEGFA levels in HUVECs treated with the specified concentrations of the respective EVs. The full-length blots are presented in Fig. S16. D, the mRNA expression levels of cyclin D1 and c-Myc were measured in HUVECs treated with 100 μg/ml corresponding EVs for 24 h. Relative gene expression was normalized to that of GAPDH, and the data were analyzed via the 2^−ΔΔCt^ method. Bars represent the mean ± SD from three independent experiments. D, statistical analysis was performed using one-way ANOVA, followed by Tukey’s multiple-comparison test. Statistical significance is indicated as ∗p < 0.05 and ∗∗p < 0.01. Alix, apoptosis-linked gene 2–interacting protein X; DAPI, 4′-6-diamidino-2-phenylindole; HUVEC, human umbilical vein endothelial cell; VEGFA, vascular endothelial growth factor A.

Discussion

Various strategies involving MSCs and their secreted EVs have been extensively explored in regenerative medicine. The components sorted into EVs may influence their functional outcomes, either beneficial or detrimental, which is closely related to the nature of the cells that secrete them. In the present study, our results revealed that Alix-mediated selective packaging of β-catenin into EVs enhances the proangiogenic function of EVs. Compared with those derived from normal hP-MSCs, those derived from Alix-OE-hP-MSCs display increased proangiogenic activity, leading to improved structural and functional recovery in a hindlimb ischemia model. In contrast, EVs from Alix-KD-hP-MSCs exhibited reduced angiogenic potential. This study highlights a new mechanism by which β-catenin, previously known for its role in cell growth and differentiation, contributes to the formation of new blood vessels (Fig. 7).Figure 7Graphical summary of the effects of Alix on angiogenesis via hP-MSC–derived EVs. Alix promotes the selective sorting of β-catenin into EVs through their interaction. Consequently, the β-catenin signaling pathway is activated, which increases the expression of angiogenesis-related genes via transcriptional mechanisms, thereby augmenting the angiogenic potency of endothelial cells. Alix, apoptosis-linked gene 2–interacting protein X; ER, endoplasmic reticulum; EV, extracellular vesicle; hP-MSC, human placenta–derived mesenchymal stem cell; ILV, intraluminal vesicle; MVB, multivesicular body; TCF, T-cell factor, transcription factor, the major end point mediators of Wnt signaling.

MSC-derived EVs contain a diverse repertoire of bioactive molecules, including proteins, mRNAs, and miRNAs, and are increasingly recognized as important mediators of intercellular signaling (21). Several miRNAs have been widely reported to regulate angiogenesis by targeting multiple downstream effectors. For example, miR-126 has been shown to suppress VEGF-A signaling and inhibit tumor angiogenesis in MCF7 cells (22), whereas in other contexts, it promotes angiogenesis by targeting PIK3R2 and modulating PI3K signaling (23). Similarly, miR-21 promotes angiogenesis by directly targeting the 3′ untranslated region of SPRY2, leading to suppression of SPRY2 expression (24). Given the broad target spectra and strong context dependence of these miRNAs, the functional consequences of their downregulation in Alix-KD-EVs are likely complex. Although reduced miR-126 and miR-21 levels may contribute to the impaired proangiogenic activity of EVs, elucidating their precise roles in EV-mediated angiogenic regulation would require more extensive mechanistic analyses.

EVs derived from hP-MSCs significantly enhance wound healing and facilitate the regeneration of skin appendages, such as hair follicles and sebaceous glands, in rat models (25, 26). β-catenin is a key protein involved in the canonical Wnt signaling pathway, which plays crucial roles in both animal development and cancer progression. Alix, an accessory protein of the ESCRT machinery, helps regulate the endosomal sorting of proteins into EVs. This study revealed that Alix specifically packages β-catenin into EVs, thereby promoting angiogenesis. Consistent with our findings, exosomes derived from Alix-OE iPSCs have been reported to promote angiogenesis (11), supporting a conserved role for Alix in modulating EV function across different stem cell types.

Alix expression is regulated by multiple internal and external factors, thereby influencing the biogenesis of EVs (9). Cells influence EV release through interactions among Alix, Syntenin1, and Syndecan1 (27, 28). In the murine heart, genetic deletion of Alix reduces plasma EV levels without affecting cardiac structure or contractile function (29). The sorting of tetraspanins CD9, CD63, and CD81 into EVs is dependent on the Alix-mediated pathway (30), where depletion of Alix leads to a reduction in the CD9 content within EVs. Although Alix regulates the sorting of EV cargo, studies suggest that it does not substantially affect the overall quantity of EVs. Consistent with our NTA, Alix deficiency did not significantly alter EV secretion or yield in HEK293 cells (10). Moreover, EVs derived from Alix-knockout iPSCs exhibit a mild increase in vesicle size (11), which aligns with our observation that EVs from Alix-KD hP-MSCs display a modest increase in average particle size and diameter. Together, these findings suggest that subtle EV enlargement may represent a common phenotypic feature associated with Alix depletion.

Since the effects of EVs depend on their molecular content, their therapeutic potential may be enhanced by selectively loading beneficial molecules. Our findings indicate that the functional changes observed in the EVs derived from Alix-modulated hP-MSCs are attributable to the role of Alix in facilitating the incorporation of β-catenin. β-catenin has been shown to interact with the ESCRT complex component CHMP4B (13), and Alix also interacts with CHMP4B (30). Notably, we demonstrated that Alix interacts with β-catenin independently of CHMP4B and that depletion of Alix markedly reduces β-catenin levels in EVs.

β-catenin is a key component of the Wnt signaling pathway. Numerous studies have shown that the Wnt/β-catenin signaling pathway regulates the expression of genes associated with angiogenesis (31, 32, 33, 34). EVs derived from human umbilical cord tissue have been shown to activate the Wnt4/β-catenin pathway, promote β-catenin nuclear translocation, and increase the expression of proliferating cell nuclear antigen and angiogenesis-related genes, such as VEGFA, thereby increasing angiogenesis (20, 35). Consistent with previous reports, EVs derived from hP-MSCs similarly promoted β-catenin nuclear translocation and activated the Wnt pathway, leading to increased expression of VEGFA and downstream components, such as cyclin D1 and c-Myc. Moreover, previous studies have reported that EVs derived from adipose tissue–derived MSCs fail to activate the AKT and ERK signaling pathways in vitro (36), which is consistent with our findings and suggests that the proangiogenic effects of Alix-dependent hP-MSC–derived EVs are predominantly mediated through Wnt/β-catenin signaling rather than through parallel angiogenic pathways.

Owing to the reduced β-catenin levels in Alix-KD hP-MSC–derived EVs, β-catenin levels in HUVECs cocultured with these EVs were also reduced, and β-catenin nuclear translocation was inhibited, which prevented Wnt pathway activation. Wnts have also been shown to colocalize with one of the tetraspanin family members, CD81, and the ESCRT-I complex component tumor susceptibility gene (Tsg101) on EVs (37, 38). Although Alix has been shown to interact with Tsg101 (39), its role in regulating Wnt sorting into EVs and in modulating subsequent Wnt signaling activation remains unclear. Further investigations will enhance our understanding of Alix and provide insights into EV cargo sorting and its functional implications.

Conclusion

In summary, EVs derived from Alix-KD-hP-MSCs attenuated the therapeutic effects on cell migration and angiogenesis. Similarly, EVs from Alix-KD-hP-MSCs showed reduced therapeutic efficacy in a hindlimb ischemia model, whereas those from Alix-OE-hP-MSCs demonstrated improved therapeutic outcomes. Alix mediates the sorting of increased amounts of β-catenin into EVs through its interaction with β-catenin, resulting in increased nuclear β-catenin levels in recipient cells. Consequently, this further activates the Wnt pathway, increasing the expression of angiogenesis-related genes and promoting angiogenesis. Taken together, the results of this study reveal a novel mechanism by which the Alix-mediated packaging of β-catenin into EVs enhances their ability to promote angiogenesis. This finding has significant implications for the development of new therapeutic strategies for treating ischemic diseases by enhancing angiogenesis.

Experimental procedures

Cell culture and transfection

hP-MSCs, purchased from AmCellGene Co Ltd, were cultured in line with previous reports (40, 41). Briefly, hP-MSCs were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin–streptomycin (Gibco). hP-MSCs from passages 3 to 9 were utilized in subsequent experiments. HUVECs, HEK293T cells (a human kidney cell line, JLCE2306), and HeLa cells (human cervical cancer cells, JLC-E2224) were procured from the American Type Culture Collection. HUVECs were cultured in EGM2 medium (Lonza). HEK293T and HeLa cells were cultured in DMEM (Gibco) supplemented with 10% FBS and 1% penicillin–streptomycin. All experiments were performed using cells confirmed to be free of mycoplasma contamination, as routinely verified by PCR testing. For transfection, cells were seeded at 70% to 80% confluence in 6-well plates, 24-well plates, or 10 cm dishes. After 24 h, the plasmids were transfected with polyethyleneimine (Polysciences) following the manufacturer’s instructions.

Plasmids and generation of Alix-KD hP-MSCs

Human Alix complementary DNA (cDNAs) were subsequently cloned and inserted into the vector pCMV-3HA (Clontech). Human β-catenin cDNAs were subsequently cloned and inserted into the vector pCE-puro-3×FLAG. The Alix-KD hP-MSCs were generated via shRNA. An shRNA targeting Alix was designed via the shRNA Sequence Designer (Clontech) and cloned and inserted into the pSIREN-RetroQ vector (Clontech). The target sequences for the Alix shRNA and control shRNA constructs were as follows: 5′-CTGCTAAACATTACCAGTTT-3′ and 5′-GAAGTAAGCGATATACATA-3′. HEK293T cells were transfected with 1 μg of pMLV-Gag-Pol, 0.5 μg of pVSV-G, or 1 μg of the pSIREN-RetroQ construct. At 48 h after transfection, the supernatants were harvested to infect hP-MSCs. After 48 h, the medium containing 0.5 mg/ml puromycin (Sigma‒Aldrich) was supplemented for selection. Western blot assays were used to analyze the efficiency of Alix expression. The KD efficiency of Alix was assessed via Western blot analysis or qRT‒PCR.

Generation of Alix-OE hP-MSCs

The oligonucleotides of the Alix mimic and its nonspecific control were amplified and cloned and inserted into pQCXIP constructs to generate an Alix oligonucleotide vector (pQCXIP-Alix) and its nonspecific control vector (control). The viral expression constructs and the packaging plasmid mixture of pMLV-Gag-Pol and pVSV-G were subsequently cotransfected into HEK293T cells. The vectors were collected from the supernatant at 48 h. hP-MSCs were then transfected with pQCXIP-Alix or the control and subcultured for 2 weeks in complete growth medium supplemented with 0.5 mg/ml puromycin to select for transduced cells.

EV isolation and characterization

To isolate EVs from the cellular supernatant, hP-MSCs were rinsed with PBS, and the medium was replaced with DMEM containing 10% EV-depleted FBS (42, 43, 44). EV-free FBS was obtained by ultracentrifugation at 100,000g for 90 min. After a 48-h incubation, the culture medium was collected, and the EVs were isolated at 4 °C by sequential centrifugation. Briefly, the medium was centrifuged at 500g for 10 min to remove the cells. The supernatant was centrifuged at 2000g for 30 min and then at 10,000g for 30 min. The resulting supernatant was filtered through a 0.22-μm filter, and the EVs were pelleted by ultracentrifugation at 100,000g (Beckman Type 32 Ti) for 120 min. Finally, the EV pellet was resuspended in Dulbecco’s PBS or lysis buffer before further analysis.

To quantify the total protein concentration of the EVs after ultracentrifugation, the concentration was measured via a BCA protein assay kit (Promega). The size of the EVs was determined via DLS measurements via a BI-200SM laser scatterer (ZetaPALS). To verify the morphology of the isolated EVs, the EV pellet was dropped onto a carbon film (Zongjing Keji Technology) and incubated at room temperature for 5 min. After negative staining with 2% phosphotungstic acid, the samples were air dried, and images were captured via TEM (Talos F200C).

Scratch wound healing assay

This assay was used to evaluate the migration capability of HUVECs. HUVECs were seeded in a 6-well plate, and when the confluence of the cells reached greater than 80%, a scratch wound was generated via the tip of a 10 μl micropipette. After the medium was replaced with fresh medium, the EVs were incubated with the cells for 24 h. Images were taken at 0 h and 24 h via a microscope (Olympus). Mobility was quantified via ImageJ software (National Institutes of Health).

Tube formation assay

Tube formation assays were conducted in 48-well plates coated with Matrigel (Corning). The Matrigel-coated plates were incubated at 4 °C overnight, followed by incubation at 37 °C for 30 min to promote gelatinization. HUVECs (3 × 10^4^ cells/well) were seeded onto Matrigel-coated plates, and EVs were coincubated with the cells for 12 h at 37 °C under 5% CO_2_. Bright-field microscopy (Olympus) was used to capture images. The total length and number of branches of the network structures in three randomly selected fields per well were quantified via ImageJ software (National Institutes of Health) to assess the proangiogenic potential of HUVECs.

Murine hindlimb ischemia models

In this study, transgenic Vegfr2-luc mice for monitoring angiogenesis were obtained from Xenogen Corporation (45, 46). The Vegfr2 promoter in mice can regulate the expression of the Fluc reporter gene. Mice (8–10 weeks old, male, weighing 25–30 g) were anesthetized with 2.5% avertin (240 mg/kg, Sigma‒Aldrich) intraperitoneally. The femoral artery was then ligated after being separated from the femoral vein and nerve, and the arterial bifurcation was also ligated near the knee. The main branch of the femoral artery was then removed between the two nodes, and the mouse hind limb ischemia model was established as described previously (19, 47). After the ischemia model was established, 100 μg of the corresponding EVs obtained from cells after different treatments and resuspended in PBS were injected intramuscularly into the ischemic hind limb in a total volume of 40 μl, and an equal volume of PBS was injected as a control. At the end of the experiment, the mice were euthanized by inhalation of an overdose of isoflurane (5%), followed by cervical dislocation. Mice in the sham group underwent a skin incision followed by immediate suturing, without any manipulation of the underlying femoral artery or adjacent neurovascular structures. All the experimental procedures for the animal studies were approved by the Institutional Animal Care and Use Committee of Nankai University and conducted in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals, eighth edition (approval no.: 20230027). The work has been reported in line with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines 2.0.

BLI analysis

The effects of EVs produced by hP-MSCs with Alix overexpression or KD on angiogenesis in mice were tracked via real-time monitoring of Vegfr2 expression in transgenic Vegfr2-luc mice. As described previously (45, 48), the mice were anesthetized with 2% isoflurane (RWD) and injected intraperitoneally with d-luciferin (150 mg/kg; Biosynth International), a substrate for Fluc, and images were captured a few minutes later via an IVIS Lumina II system (Xenogen Corporation). BLI signals were obtained by measuring the mean radiance of regions of interest. For in vivo experiments, regions of interest were selected by covering the injection area. VEGFR2 expression was visualized via BLI.

IF staining

The tissue samples stored at −80 °C were cut into 6-μm-thick sections. The frozen sections were blocked and incubated with primary antibodies against CD31 (rat anti-mouse, Abcam) overnight at 4 °C. The sections were incubated with the corresponding fluorescently labeled secondary antibodies (Life Technologies) for 2 h. The cell nuclei were stained with 4′-6-diamidino-2-phenylindole (Vector Laboratories). The slides were mounted with a mounting solution containing an antifade agent. To quantify the IF staining of the tissues, the number of capillaries in three random fields of view in each group was measured via ImageJ software.

RT‒qPCR analysis

Total RNA was extracted with TRIzol reagent (Invitrogen Corp) following the manufacturer’s protocol. BioScript All-in-One cDNA Synthesis SuperMix (Bimake) was used to reverse transcribe the total RNA into cDNA. RT‒qPCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems). GAPDH was used as an internal control. Relative gene expression fold changes were measured in triplicate and calculated via the 2^-ΔΔCT^ method. The primers used in this study are shown in Table S1.

IF assay

Cells were seeded in 24-well plates coated with coverslips and transfected with the corresponding plasmids. After 48 h, the cells were fixed with 4% formaldehyde for 20 min and then treated with PBS containing 0.1% Triton X-100 for 10 min to perforate the cell membrane surface. Next, 5% skim milk and 5% BSA in PBS were added to the wells and incubated at room temperature for 2 h or at 4 °C overnight to block contaminants. The cells on coverslips were incubated with primary antibodies at 4 °C overnight, washed with PBS, and incubated with secondary antibodies conjugated with fluorescent dyes in the dark for 2 h. The coverslips were washed again with PBS, and then, 4′-6-diamidino-2-phenylindole was added to the wells and incubated in the dark for 10 min. After washing with PBS, the coverslips were mounted on slides with glycerol containing a radioquencher and allowed to dry at room temperature in the dark. Images were captured under a confocal fluorescence microscope (Leica TCS SP5; Wetzlar). Anti-HA (1:100 dilution, 51064-2-AP), anti-FLAG (1:100 dilution, 66008-4-Ig), and anti-β-catenin (1:100 dilution, 51067-2-AP) primary antibodies, along with their corresponding secondary antibodies, were all purchased from Proteintech.

Separation of the cell nucleus and cell cytoplasm

The cells were collected, washed with PBS, centrifuged, resuspended in 500 μl of buffer A (containing protease inhibitors, 1 M Hepes, 2 M KCl, 1 M MgCl_2_, and 1 M DTT), lysed on ice for 30 min, pipetted and aspirated 10 times with a 1 ml syringe (26 G) needle, placed on ice for 15 min, and centrifuged at 2800 rpm for 5 min at 4 °C. The supernatant and pellet were collected. The supernatant contained the cytoplasm, and the pellet contained the nuclei. The pellet was further resuspended in 1 ml of solution I (0.25 M sucrose, 10 mM MgCl_2_, and protease inhibitors), and 3 ml of solution II (0.35 M sucrose, 0.5 mM MgCl_2_, and protease inhibitors) was added to the resuspended pellet. A cleaner nuclear pellet was obtained after centrifugation at 3900 rpm for 5 min at 4 °C. The nuclear pellet was resuspended in a certain amount of buffer A. The collected samples were stored at −80 °C and then subjected to immunoblot analysis.

Western blot analysis

The cells or EV pellets were harvested, lysed with radioimmunoprecipitation assay buffer (Solarbio) on ice for 40 min, and then terminated with protein loading buffer containing 2% SDS. The total protein from the EV pellets and cell lysates was boiled at 100 °C for 20 min and then subjected to SDS-PAGE (10% polyacrylamide). The proteins were transferred to polyvinylidene difluoride membranes (Millipore). After being blocked with 5% nonfat milk for 45 min, the membranes were incubated with primary antibodies overnight at 4 °C and then for 45 min at room temperature with the appropriate secondary antibodies. Finally, the substrate for horseradish peroxidase was added to the membrane for 2 min, and the immunoreactive proteins were detected by chemiluminescence (Merck Millipore). The following primary antibodies were employed for Western blot analysis: rabbit anti-Alix (1:1000 dilution, ab275377, Abcam), rabbit anti-β-catenin (1:1000 dilution, ab32572, Abcam), mouse anti-CD9 (1:500 dilution, sc-20048, Santa Cruz), rabbit anti-GM130 (1:1000 dilution, ab52649, Abcam), rabbit anti-VEGFA (1:500 dilution, 81323-2-RR, Proteintech), rabbit anti-GSK3β (1:1000 dilution, 82061-1-RR, Proteintech), rabbit anti-phospho-GSK3β (1:1000 dilution, BM4837, BOSTER), rabbit anti-Histone H3 (1:1000 dilution, ab1791, Abcam), mouse anti-GAPDH (1:3000 dilution, 60004-1-Ig, Proteintech), mouse anti-β-actin (1:2000 dilution, 66009-1-Ig, Proteintech), mouse anti-FLAG (1:3000 dilution, 66008-4-Ig, Proteintech), and rabbit anti-HA (1:3000 dilution, 81290-1-RR, Proteintech). The aforementioned antibodies were used following the manufacturer’s instructions. The secondary antibodies were purchased from Proteintech. The specificity of the primary antibodies was validated by comparing signals in Ctrl-KD and Alix-KD cells.

Statistical analysis

All the data are from at least three independent experiments. The data are expressed as the mean ± SD. Statistical analysis was performed via Student's t test or one- or two-way ANOVA via GraphPad Prism (GraphPad Software, Inc). Differences were considered statistically significant when p values were less than 0.05.

Data availability

All data are included in the article and its supporting information files or are available from the corresponding authors upon reasonable request.

Supporting information

This article contains supporting information.

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.