Original Article

Apoptotic vesicles activate autophagy

in recipient cells to induce angiogenesis
and dental pulp regeneration
Zihan Li,1,2,6 Meiling Wu,1,6 Siying Liu,2,6 Xuemei Liu,1,2 Yu Huan,3 Qingyuan Ye,2,4 Xiaoxue Yang,1 Hao Guo,1
Anqi Liu,1 Xiaoyao Huang,1 Xiaoshan Yang,2,5 Feng Ding,2 Haokun Xu,2 Jun Zhou,2 Peisheng Liu,1 Shiyu Liu,2
Yan Jin,2 and Kun Xuan1
1State Key Laboratory of Military Stomatology and National Clinical Research Center for Oral Diseases and Shaanxi Clinical Research Center for Oral Diseases, Department
of Preventive Dentistry, School of Stomatology, The Fourth Military Medical University, 145West Changle Road, Xi’an, Shaanxi, China; 2State Key Laboratory of Military
Stomatology and National Clinical Research Center for Oral Diseases and Shaanxi International Joint Research Center for Oral Diseases, Center for Tissue Engineering,
School of Stomatology, The Fourth Military Medical University, 145West Changle Road, Xi’an, Shaanxi, China; 3Department of Neurosurgery, Xijing Hospital, Air
Force Medical University, Xi’an, Shaanxi, China; 4State Key Laboratory of Military Stomatology and National Clinical Research Center for Oral Diseases and Shaanxi
Engineering Research Center for Dental Materials and Advanced Manufacture, Department of Periodontology, School of Stomatology, The Fourth Military Medical
University, Xi’an, Shaanxi, China; 5Stomatology Hospital, Southern Medical University, Guangzhou, Guangdong, China

Extracellular vesicles (EVs) derived from living cells play medical need despite the remarkable effectiveness of regenerative
important roles in donor cell-induced recipient tissue regener- medicine of a variety of tissues.
ation. Although numerous studies have found that cells un-
dergo apoptosis after implantation in an ischemic-hypoxic Mesenchymal stem cell (MSC) transplantation has been confirmed to
environment, the roles played by the EVs released by apoptotic have beneficial effects by activating the angiogenic function of host
cells are largely unknown. In this study, we obtained apoptotic ECs and achieving regeneration efficiency in ischemic tissues.6 Recent
vesicles (apoVs) derived from human deciduous pulp stem cells reports have shown that the ischemic-hypoxic environment activates
and explored their effects on the dental pulp regeneration pro- the angiogenic potential of MSCs, including their ability to release
cess. Our work showed that apoVs were ingested by endothelial angiogenic factors,7 secrete exosomes,8 and alter EC behavior.9
cells (ECs) and elevated the expression of angiogenesis-related However, the ischemic-hypoxic environment usually leads to the
genes, leading to pulp revascularization and tissue regenera- apoptosis of the donor cells,10 which is not conducive to favorable
tion. Furthermore, we found that, at the molecular level, revascularization or effective regeneration. Moreover, whether
apoV-carried mitochondrial Tu translation elongation factor apoptosis influences angiogenesis is largely unclear, and the potential
was transported and regulated the angiogenic activation of mechanisms are unknown.
ECs via the transcription factor EB-autophagy pathway. In a
beagle model of dental pulp regeneration in situ, apoVs re- Apoptosis is an autonomously regulated cellular end-of-life process
cruited endogenous ECs and facilitated the formation of and is considered to be a passive phenomenon.11 This view is
dental-pulp-like tissue rich in blood vessels. These findings re- beginning to change, as studies have shown that apoptosis plays an
vealed the significance of apoptosis in tissue regeneration and important role in regulating tissue homeostasis and promoting tissue
demonstrated the potential of using apoVs to promote angio-
genesis in clinical applications.
Received 14 December 2021; accepted 7 May 2022;
https://doi.org/10.1016/j.ymthe.2022.05.006.
6
These authors contributed equally
Correspondence: Shiyu Liu, PhD, Research and Development Center for Tissue
INTRODUCTION Engineering, School of Stomatology, The Fourth Military Medical University,
A well-functioning blood vascular system is essential to tissue regen- 145West Changle Road, Xi’an, Shaanxi 710032, China.
eration, and in transplantation, it participates in gas exchange, E-mail: liushiyu@vip.163.com
Correspondence: Yan Jin, PhD, Research and Development Center for Tissue
nutrient transport, and waste clearance between the donor and host Engineering, School of Stomatology, The Fourth Military Medical University,
tissues.1 However, endogenous endothelial cells (ECs) seldom prolif- 145West Changle Road, Xi’an, Shaanxi 710032, China.
erate, remaining in an inactive state for decades in adult life,2,3 which E-mail: yanjin@fmmu.edu.cn
Correspondence: Kun Xuan, PhD, Department of Preventive Dentistry, School of
results in slow and inefficient revascularization, and ultimately, tis-
Stomatology, The Fourth Military Medical University, 145West Changle Road,
sues fail to regenerate,4 especially ischemic-hypoxic tissues.5 Thus, Xi’an, Shaanxi 710032, China.
revascularization of ischemic-hypoxic tissue remains an unmet E-mail: xuankun@fmmu.edu.cn

Molecular Therapy Vol. 30 No 10 October 2022 ª 2022 The Author(s). 3193

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
 Molecular Therapy

repair and regeneration.12,13 Apoptotic cells can release a variety of (Figure S1B). Crystalline violet staining showed that hDPSCs
substances that regulate body homeostasis, including metabolites generated single colony clusters (Figure S1C).
and extracellular vesicles (EVs). It has been reported that these spe-
cific metabolites can act as good-bye signals to actively modulate Then, we prepared human tooth scaffolds combined with aggregated
the biological functions of neighboring cells.14,15 Moreover, apoptotic hDPSCs (the hDPSC group) or PBS gel (the control [Ctrl] group) and
vesicles (apoVs), types of EVs, can also transfer multifarious sub- implanted them into the dorsum of athymic nude mice (Figures 1A
stances, including microRNAs (miRNAs), proteins, and lipids, that and 1B). After 2 months, hematoxylin-eosin ( H&E) staining
initiate signal transduction.16 EVs have been reported to promote showed the formation of dental-pulp-like tissue in the hDPSC group
angiogenesis during the regeneration process.17 However, the (Figure 1C). To verify the H&E histology, we performed immunoflu-
mechanisms through which apoVs influence angiogenesis and orescence staining, which also showed the presence of dentin
tissue regeneration remain unclear. sialophosphoprotein (DSPP)- and dentin matrix protein 1 (DMP1)-
positive odontoblast layers in newly formed tissue, which was similar
In recent years, MSC transplantation has led to significant outcomes to the natural dental pulp structure (Figure 1D).
in the field of maxillofacial regeneration.18,19 We previously showed
that aggregated human deciduous pulp stem cell (hDPSC) transplan- To elucidate whether these cells undergo apoptosis, aggregated
tation enabled the morphological and functional regeneration of full- hDPSCs were implanted in an ischemic-hypoxic environment.
length dental pulp, but the underlying mechanism remains unclear.20 TUNEL staining results showed that a significant portion of the
Dental pulp is a vascular-rich tissue in the stable root canal, which is aggregated hDPSCs underwent apoptosis 24, 48, or 72 h after implan-
connected to surrounding tissue only through the apical foramen;21 tation (Figure 1E). We also validated this result using flow cytometry
this special structure provides a powerful model to discover the signif- and found that the ratio of apoptotic cells among the aggregated
icance of apoptosis and explore the function of apoVs in vasculariza- cells reached 47% within 72 h (Figure 1F). These data demonstrate
tion and tissue regeneration. Moreover, translational application of that aggregated hDPSCs underwent apoptosis shortly after
apoVs may help establish a potential noncellular treatment for tissue implantation.
regeneration in an ischemic-hypoxic environment.
Apoptosis of aggregated hDPSCs was necessary for dental pulp
In this study, we first explored the effect of hDPSC apoptosis on regeneration
dental pulp regeneration. Then, we prepared apoptotic vesicles To explore the role played by apoptosis in dental pulp regeneration,
derived from hDPSCs (hDPSC-apoVs) and tested their proangio- we pretreated aggregated hDPSCs with Z-VAD, an apoptosis
genic effect on ECs. Then, we tested the ability of apoVs to stimulate inhibitor, and the inhibition efficiency was verified by TUNEL stain-
angiogenesis and improve blood supply to the root canal. Mechanis- ing (Figure S2D). We implanted tooth scaffolds filled with aggregated
tically, we demonstrated that hDPSC-apoVs promoted EC autophagy hDPSCs (the hDPSC group) or aggregated hDPSC pretreated with
by transferring mitochondrial Tu translation elongation factor Z-VAD (the Z-VAD group) subcutaneously in the dorsum of athymic
(TUFM). Furthermore, autophagy signaling activated the angiogenic nude mice. After 2 months, H&E staining results showed that the
potential of ECs. Taken together, our findings indicated that apoVs mice in the hDPSC group formed good dental-pulp-like tissues, as
can activate autophagy of recipient cells to induce host blood vessels expected (Figure 2A). In contrast, the Z-VAD group mice failed to
to grow in ischemic-hypoxic environments, providing additional achieve dental pulp regeneration after apoptosis inhibition. In
insights into improvements for tissue regeneration. addition, the donor cells underwent necrosis due to insufficient blood
supply (Figure 2B). These results suggest that apoptosis was necessary
RESULTS for aggregated hDPSC-induced dental pulp regeneration.
Aggregated hDPSCs undergo apoptosis that leads to dental
pulp regeneration Stem cells can release an abundance of EVs that contain functional
Dental pulp is crucial for maintaining tooth vitality. However, its elements that are useful for tissue regeneration.17 The role played
limited self-repair capacity makes dental pulp vulnerable to necrosis by apoVs released by apoptotic cells during dental pulp regeneration
by trauma and infections.22 In addition, the restriction of blood deserves further investigation. We induced hDPSC apoptosis with
supply through the narrow apical foramen makes regeneration STS, a protein kinase C inhibitor (Figure S1D). Next, we isolated
particularly difficult, which leads to the apoptosis of donor cells.23 apoVs from apoptotic hDPSCs using a gradient centrifugation proto-
We investigated this outcome using an athymic nude mouse model col (Figure S2A) and characterized apoV morphology (Figures 2C,
(the details are presented in the materials and methods section). 2D, and S2C). The size distribution of the apoVs was tested by dy-
namic light scattering (Figure 2E). Then, fluorescence staining and
First, we characterized the hDPSCs used in this study. Flow cytometry flow cytometry were performed to detect the exposure of phosphati-
results showed that hDPSCs highly expressed CD105, CD73, and dylserine (PtdSer), a specific marker of apoVs (Figures 2F and S2B). A
CD90 but not CD45, CD34, CD14, CD19, and HAL-DR (Figure S1A). western blot analysis showed that apoVs expressed a high level of
Alizarin red staining and oil red O (ORO) staining indicated that cleaved caspase-3 (Figure 2G). These results indicated the effective
these cells exhibited osteogenic and lipogenic differentiation potential extraction of apoVs.

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Figure 1. Aggregated hDPSCs undergo apoptosis shortly after implantation

(A) Preparation of implanted tooth scaffold, with a height of 5 mm. (B) Schematic of the tooth transplantation process in the dorsum of athymic nude mice. Tooth scaffolds
were filled with aggregated hDPSC. (C) H&E staining shows the regeneration of dental-pulp-like tissue; dp, dental pulp; d, dentin. Scale bars, 100 mm in low-magnification
images and 50 mm in high-magnification images. (D) Immunofluorescence analysis shows the expression of odontogenic differentiation markers DSPP and DMP1, with
positive staining represented by green and red stains. DSPP, dentin sialophosphoprotein; DMP1, dentin matrix protein 1; d, dentin. Scale bars, 100 mm. (E) TUNEL staining
shows the apoptosis ratio of aggregated hDPSCs before transplantation (Ctrl), or 24, 48, or 72 h after transplantation. Scale bars, 100 mm in low-magnification images and
50 mm in high-magnification images; n = 5 per group. (F) Flow cytometry analysis shows the apoptosis ratio of aggregated hDPSC before transplantation (Ctrl), or 24, 48, 72,
or 96 h after transplantation. *Annexin V (+) and 7-AAD (–) cell ratio compared with Ctrl; ^annexin V (+) cells ratio compared with Ctrl; n = 3 per group. Data are presented as
mean ± SD. Statistical analyses were performed by one-way ANOVA with Tukey’s post hoc test or Welch’s ANOVA with Games-Howell post hoc test. *p < 0.05,
**/^^p < 0.01, ***p < 0.001; ns, p > 0.05.

Then, we inserted aggregated hDPSCs pretreated with Z-VAD (the regeneration and that apoVs can promote the formation of blood
Z-VAD group), aggregated hDPSCs supplemented with hDPSC- vessels.
apoVs (the hDPSC + apoV group), and Z-VAD-pretreated aggre-
gated hDPSCs supplemented with hDPSC-apoVs (the Z-VAD + hDPSC-apoVs promote tissue regeneration
apoV group) into the tooth scaffolds. Two months after in vivo im- We further explored whether apoVs individually promote blood
plantation in the dorsum of athymic nude mice, H&E staining vessel regeneration in the root-canal space. Scaffolds filled with
showed a reduced necrotic area and angiogenesis near the opening PBS gel (the PBS group) or hDPSC-apoVs gel (the apoV group)
of the tooth scaffolds in the Z-VAD + apoV group compared with were implanted into the dorsum of athymic nude mice. After
those in the Z-VAD group (Figures 2H, 2I, and 2K. Compared with 2 months, H&E staining showed the formation of dental-pulp-like
the effect on the hDPSC group, supplementation with apoVs induced tissue in the apoV group relative to the loosely aligned connective
angiogenesis in the newly formed dental-pulp-like tissue (Figures 2A tissue in the PBS group (Figures 3A and 3B [i and ii]). Additionally,
and 2J). We then labeled CD31 in blood vessels. Confocal microscopy a significant increase in de novo dentin-like tissue was found in the
images showed increased vascular density in the groups treated with apoV group through H&E and Masson’s staining (demarcated by
apoVs (Figure 2K). Collectively, these results provide strong evidence dashed yellow lines in Figures 3A and 3B [iii and iv]). Concomi-
indicating that hDPSC apoptosis plays an active role in dental pulp tantly, higher vessel density was found in the apoV group

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Figure 2. Apoptosis of hDPSCs after implantation promote the regeneration of dental pulp
(Ai and Bi) Representative microscope images of H&E staining show regeneration of dental pulp in tooth scaffolds. Scale bars, 0.5 mm. (Aii and iii and Bii and iii) Enlarged view
of boxed area in (i). Scale bars, 100 mm. (A) Regularly prepared aggregated hDPSCs. (B) Aggregated hDPSCs pretreated with Z-VAD. (C–G) Characterization of hDPSC-
apoVs. (C) Representative SEM image of apoVs. Scale bar, 200 nm. (D) Representative TEM image of apoVs. Scale bar, 500 nm. (E) Size distribution of apoVs. (F) Annexin
V staining of apoVs. Scale bars, 200 mm. (G) Protein characterization of apoVs. (Hi–Ji) Representative microscope images of H&E staining show regeneration of dental pulp in
Z-VAD group, Z-VAD + apoV group, and hDPSC + apoV. Scale bars, 0.5 mm. (Hii and iii–Jii and iii) Enlarged view of boxed area in (i). Scale bars, 100 mm. (H) Aggregated

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(Figures 3A and 3B [v]), which suggested that apoVs promoted In addition, we assessed the effect of apoVs on EC proliferation by
blood vessel regeneration in vivo. performing a MKI67/Ki67 assay, and the results showed that the
percentage of Ki67-positive cells significantly increased after apoV
Considering the favorable angiogenesis outcome induced by apoVs, treatment, indicating that apoVs promoted EC proliferation (Fig-
we further investigated the angiogenic effects of apoVs and compared ure 4G). Thus, these data indicated that the internalization of
the effects with the degree of dental pulp revascularization, which is a hDPSC-apoVs directly enhanced the angiogenic capacity of the ECs
novel clinical therapy. We constructed a preclinical large-animal in vitro.
(dog) model (the details are presented in Materials and methods),
and dental pulp in the incisors (a total of 36 teeth in 6 dogs) was ex- hDPSC-apoVs regulate EC function by inducing autophagy
tracted with a mechanical method. The revascularization treatment EVs regulate receptor cell function via the transfer of proteins, lipids,
(the Revas group) or apoV gel treatment (the apoV group) was per- and nucleic acids.24 Therefore, we identified the proteomic composi-
formed on the incisors, and the results were compared with those tions of hDPSCs and apoVs by using mass spectrometry. A total
obtained with a PBS gel (the PBS group), which was the control. Three of 9,424 proteins were identified in hDPSCs and apoVs. Among
months after treatment, H&E staining showed that no fibrous the 1,102 differentially expressed proteins between the hDPSCs
connective tissue had formed in the PBS group (Figure 3D [i]), which and apoVs, 129 were significantly upregulated in the apoVs
verified dental pulp ablation. (Figures S3A and S3B). We then focused on the pathway enriched
with the upregulated proteins by performing a Kyoto Encyclopedia
Cone-beam computed tomography (CBCT) images of the incisors of Genes and Genomes (KEGG) pathway analysis; the results
were taken, and 3D images were reconstructed before and 3 months showed that the proteins were enriched in autophagy-associated
after treatment. Compared with that in the PBS group, the dentin pathways, such as the “mTOR-signaling pathway,” “mitophagy-ani-
thickness increased by an average of 0.2 cm in the apoV group, but mal,” and “insulin-signaling pathway” (Figure S3E). These results
the increase in the Revas groups was not statistically significant indicated that hDPSC-apoVs contained many proteins that were
(Figure 3C), which demonstrated that apoVs promoted the highly related to autophagy, which might exhibit regulatory functions
development of young permanent teeth. in ECs.

Dental-pulp-like tissue contained newly formed blood vessels in the Therefore, we detected the autophagy level in ECs after apoV treat-
apoV group (Figure 3F), but after the revascularization treatment, ment. Western blot results showed that the expression of auto-
only the formation of some amorphous matrixes was observed phagy-associated proteins (ATG7, Beclin-1, and LC3) were upregu-
(Figure 3E). Immunofluorescence staining ultimately showed lated (Figure 5A). In addition, we infected ECs with adenoviruses
CD31-positive cells arranged in lumen-like shapes in the apoV group expressing mRFP-GFP-LC3 to inhibit the change in autophagic flux
(Figure 3G). Together, these results demonstrated that hDPSC-apoVs in the intracellular space. Confocal microscopy images showed the
promoted dental pulp regeneration by stimulating angiogenesis. formation of autophagosomes (both mRFP and GFP fluorescence
emitted a yellow signal) and autolysosomes (only red RFP fluores-
hDPSC-apoVs are internalized by ECs and enhance their cence was emitted) after apoV treatment. By blocking the fusion of
angiogenic capacities autophagosomes with lysosomes by treating cells with chloroquine
To investigate how hDPSC-apoVs promote angiogenesis, we labeled (CQ), we observed the accumulation of autophagosomes in the
apoVs with PKH26 and cocultured them with human umbilical vein apoV group over time (Figure 5B). Similarly, a western blot analysis
ECs for 8 h. Confocal microscopy images showed that the apoVs also showed increased expression of LC3 II (microtubule-associated
localized within ECs (Figure 4A). A western blot analysis showed protein 1 light-chain 3 II) 4, 12, or 24 h after treatment of ECs with
that the expression of angiogenesis- and cell migration-associated CQ (Figure 5C). Taken together, these data demonstrate that apoVs
proteins (HIF-1a, VEGF, ANG2, and MMP2) in ECs were signifi- activate EC autophagy.
cantly upregulated after treatment (Figure 4B). We next investigated
the effect of apoVs on EC functions. Autophagy is known to regulate homeostasis during angiogenesis and
to increase the vascular system.25 To determine whether autophagy
A Matrigel assay showed that 20 mg/mL apoVs significantly promoted performs a similar function in apoV-mediated angiogenesis, we
EC tube formation, and the tube formation ability of these cells per- treated ECs with 3-methyladenine (3-MA), an autophagy inhibitor,
sisted for a long time (Figures 4C–4E). Furthermore, a Transwell and observed the change in EC angiogenic activities after apoV treat-
assay showed that the number of migrated ECs was increased after ment. As expected, the angiogenic, migratory, and proliferative
apoVs treatment in a concentration-dependent manner (Figure 4F). functions of the ECs were reduced after autophagy inhibition

hDPSCs pretreated with Z-VAD. (I) Aggregated hDPSCs pretreated with Z-VAD supplemented with apoVs. (J) Regularly prepared aggregated hDPSCs supplemented with
apoVs. (K) Immunofluorescence analysis shows the vessel density of regenerated dental pulp tissue. Scale bars, 200 mm in low-magnification images and 50 mm in high-
magnification images; n = 5 per group. Data are presented as mean ± SD. Statistical analyses are performed by one-way ANOVA with Tukey’s post hoc test or Welch’s
ANOVA with Games-Howell post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001; ns, p > 0.05.

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Figure 3. hDPSC-apoVs induce angiogenesis and dental pulp regeneration in vivo

(AI and Bi) Representative microscope images of H&E staining show regeneration of dental pulp in tooth scaffolds. Scale bars, 1 mm. (Aii and iii and Bii and iii) Enlarged view of
boxed area in (i). Scale bars, 100 mm in low-magnification images and 50 mm in high-magnification images. (Aiv and Biv) Masson’s Trichrome staining of boxed area in (i).
(Av and Bv) Immunofluorescence analysis shows the vessel density in new-formation dental-pulp-like tissues. Scale bars, 100 mm in low-magnification images and 50 mm in
high-magnification images; n=5 per group. (C) Representative CBCT images (top) of incisor teeth before and after treatment and 3D images (bottom) of incisor teeth before
and after treatment. The amount of dentin was increased after treatment in the apoV and normal groups (white arrows); n = 5 per group. (Di and Fi) Representative mi-
croscope images of H&E staining show regeneration effect of dental pulp in beagle’s incisor teeth. Scale bars, 0.5 mm. H&E and Masson’s Trichrome staining of PBS group
(Dii and iii), dental pulp revascularization (Revas) group (Eii and iii), and apoVs group (Fii and iii). Scale bars, 100 mm in low-magnification images and 50 mm in high-
magnification images. (G) Immunofluorescence analysis shows the expression of blood vessel marker CD31, with positive represented by green stains. Scale bars, 200 mm in
low-magnification images and 50 mm in high-magnification images. Apex, root tip of teeth; bv, blood vessels; dp, dental pulp; nd, newly formed dentin; d, dentin; n = 5 per
group. Data are presented as mean ± SD. Statistical analyses were performed by Student’s t test (two-tailed) for two group comparisons and one-way ANOVA with Tukey’s
post hoc test or Welch’s ANOVA with Games-Howell post hoc test for multiple group comparisons. *p < 0.05, **p < 0.01, ***p < 0.001; ns, p > 0.05.

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(Figures 5D–5G). All these results revealed that hDPSC-apoVs hDPSC apoptosis ameliorates revascularization via autophagy
enhanced the EC angiogenic function via autophagy regulation. in vivo
To verify that autophagy was necessary for apoV-mediated EC angio-
TUFM activates EC autophagy via TFEB nuclear translocation genesis in vivo, we injected 3-MA into athymic nude mice to inhibit
Notably, previous studies have elucidated that TUFM is an autophagy autophagy and then detected the expression of LC3 to determine the
effector.26 Therefore, we sought to determine whether TUFM derived inhibition efficiency (Figure 7A). Then, we implanted tooth scaffolds
from apoVs mediates autophagy in ECs. First, a western blot analysis filled with apoVs (the 3-MA + apoV group) subcutaneously in the
was performed to verify the existence of TUFM in apoVs (Figure 6A). dorsum of autophagy-inhibited athymic nude mice. H&E staining
Then, we transfected hDPSCs with short interfering RNA (siRNA) showed that apoVs did not induce the formation of blood vessels after
against TUFM (si-TUFM) to downregulate the expression of autophagy inhibition (Figures 7B–7D). Thus, we considered that
TUFM in the apoVs (Figure 6A). Next, to determine the effects of autophagy was required for apoV-mediated angiogenesis in vivo.
TUFM on EC autophagy, we treated the ECs with two kinds of apoVs
(apoVs and si-TUFM-apoVs), and unstimulated ECs comprised the Taken together, our results led us to conclude that exogenous aggre-
control group. The results showed that the expression level of gated hDPSCs partially undergo apoptosis after implantation into the
autophagy-associated genes was distinctly downregulated in the si- root canal. apoVs derived from hDPSCs specifically activated endog-
TUFM-apoV group (Figure 6B). Finally, we found that EC tube enous EC autophagy by transferring TUFM. Furthermore, autophagy
formation, migration, and proliferation were attenuated by downre- signaling mediated EC biological behavior and caused angiogenesis.
gulating the expression of TUFM in apoVs (Figures 6C–6E). These Eventually, the accelerated revascularization promoted the efficient
results indicated that TUFM derived from hDPSC-apoVs activated regeneration of dental pulp. Significantly, the large-animal experi-
EC autophagy to regulate angiogenic capacity. ment verified the angiogenic function of the apoVs administered
independently. Overall, these results identify an unreported
Several studies have confirmed that TUFM can regulate the TFEB- mechanism of apoptosis in dental pulp regeneration (Figure 8).
induced autophagy–lysosome pathway.27 To investigate whether
TUFM derived from hDPSC-apoVs activates EC autophagy in the DISCUSSION
same way that it induces TFEB-induced autophagy, we first labeled As determined by their biogenesis, EVs can be divided into three
lysosomes to monitor lysosomal biogenesis. LysoTracker staining main categories: exosomes, microvesicles, and apoVs. Formerly, the
showed that the number of lysosomes was increased after apoV products of apoptotic cells were considered waste or deleterious
treatment, similar to the effect caused by Torin 1, a lysosomal acti- substances.11 Recently, mounting evidence has suggested that apoVs
vator (Figure 6F). Moreover, the expression of lysosomal-associated play key roles not only in the regulation of normal physiological pro-
proteins (LAMP1 and CLCN7) and TFEB was upregulated cesses, such as spermatogenesis,28 renewal of the outer segment of
(Figure 6G). photoreceptors,29 and immune surveillance30 but also in tissue repair
and regeneration.12,13 Our study confirmed that apoVs released by
TFEB regulates the expression of autophagy-related genes by moving exogenous hDPSCs exerted positive effects on revascularization dur-
to the nucleus. We evaluated the content variations of TFEB in the ing stem-cell-mediated pulp regeneration and that apoV-loaded
nucleus after apoVs treatment. In the apoV group, TFEB expression TUFM was transported to regulate the activation of endogenous
in the nucleus was increased (Figure 6H), and TFEB nuclear translo- ECs via the TFEB-induced autophagy pathway. Previous studies
cation was observed by immunofluorescence (Figure 6I). Then, by have shown that autophagy is a double-edged sword during angio-
increasing or decreasing TFEB expression levels in ECs, we confirmed genesis and that the precise role played by autophagy in various
that TFEB regulated autophagy. (Figures 6J and 6K). We further organismic microenvironments might substantially differ. Our
explored the effect of TFEB on EC angiogenic capacity. After TFEB findings revealed that autophagy activated by hDPSC-apoVs pro-
knockdown, the tube formation, migration, and proliferation func- moted the angiogenic abilities of ECs, including their proliferation,
tions of the ECs induced by apoVs were attenuated (Figures 6L– migration, differentiation, and secretion. These findings facilitate
6N). Collectively, these results revealed that TUFM derived from our understanding of apoptosis in early tissue regeneration
hDPSC-apoVs activated EC autophagy and promoted EC angiogen- after stem cell transplantation and provide a reference for future
esis via the TFEB-induced autophagy-lysosome pathway. studies.

Figure 4. hDPSC-apoVs enhance the angiogenic capacity of the ECs

(A) Representative confocal orthogonal view shows the uptake of PKH26-labeled apoVs (red) by ECs (green), counterstained with Hoechst (blue). Scale bar, 50 mm. (B)
Western blot analysis shows the expressions of HIF-1a, VEGF, ANG2, and MMP2 in ECs after incubation with apoVs at concentrations of 10, 20, or 30 mg/mL for 12 h. (C–E)
Matrigel analysis shows the effect of hDPSC-apoVs on tube formation capacity of ECs. Scale bar, 100 mm. (D) *Comparison between Ctrl and 20 mg/mL, (E) *compared with
Ctrl. (F) Transwell assay shows the effect of hDPSC-apoVs on migration capacity of ECs. Scale bar, 100 mm. (G) Immunofluorescent staining of MKI67 shows the effect of
hDPSC-apoVs on the proliferation capacity of ECs. Scale bar, 100 mm; n = 5 per group. Data are presented as mean ± SD. Statistical analyses were performed by Student’s t
test (two-tailed) or Student’s t test with Welch correction (two-tailed) for two-group comparisons and one-way ANOVA with Tukey’s post hoc test or Welch’s ANOVA with
Games-Howell post hoc test for multiple-group comparisons. *p < 0.05, **p < 0.01, ***p < 0.001; ns, p > 0.05.

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Figure 5. hDPSC-apoVs activate the autophagy of ECs to induce angiogenesis

(A) Western blot analysis shows autophagy-associated genes (ATG7, Beclin-1, and LC3) expression in ECs after incubation with apoVs at concentrations of 10, 20, or
30 mg/mL. (B) Representative confocal microscopy images show the numbers of autophagosomes (yellow) and autolysosomes (red) per cell. Scale bars, 10 mm. (C) Expres-
sion of LC3 in ECs cocultured with 20 mg/mL apoVs after treatment with or without CQ was evaluated by western blots at 0, 4, 12, or 24 h. After pretreatment with or without
3-MA, the angiogenic capacity of ECs, including tube formation (D and E), migration (F), and proliferation (G) were detected following coculture with apoVs (20 mg/mL). Scale
bar, 100 mm; n = 5 per group. Data are presented as mean ± SD. Statistical analyses were performed by one-way ANOVA with Tukey’s post hoc test or Welch’s ANOVA with
Games-Howell post hoc test for multiple-group comparisons. *p < 0.05, **p < 0.01, ***p < 0.001; ns, p > 0.05.

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Figure 6. TUFM derived from hDPSC-apoVs activate EC autophagy and promote EC angiogenesis via the TFEB-induced autophagy-lysosome pathway
(A) Western blotting analysis shows the presence of TUFM in apoVs derived from hDPSCs, si-NC-treated hDPSCs, and si-TUFM-treated hDPSCs. (B) Western blot analysis
shows autophagy-associated gene (ATG7, Beclin-1, and LC3) expressions in ECs after incubation with apoVs or si-TUFM-apoVs. The angiogenic capacity of ECs, including
tube formation (C), migration (D), and proliferation (E) were detected after treatment with apoVs or si-TUFM-apoVs. Scale bar, 100 mm. (F) Representative confocal
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Revascularization of ischemic-hypoxic tissues remains an unmet need MATERIALS AND METHODS

in clinical treatment. Angiogenesis causes vessel sprouting from pre- Isolation, culture, and characterization of the hDPSCs
existing vessels. Attracted by proangiogenic signals, ECs migrate to The use of dental pulp from human deciduous teeth was approved by
form blood vessels.31 As the angiogenic effect of apoVs was confirmed, the Ethics Committee of the Fourth Military Medical University, and
cell apoptosis and apoV release appeared to be involved in a self-reg- informed consent was obtained from the donors. hDPSCs were isolated
ulatory process of the aggregated hDPSCs that improved the ischemic- and cultured from the deciduous teeth using the collagenase digestion
hypoxic environment in the root canal. Our results may explain the method, as previously reported.35 When the primary hDPSCs prolifer-
relationship between a certain degree of donor cell apoptosis and ated to 80%–90% confluence, the adherent cells were digested with
the beneficial efficiency of apoptosis in tissue regeneration. Our find- 0.25% trypsin (MP Biomedicals, USA) and passaged in vitro. Third-
ings revealed the underlying mechanisms of stem-cell-mediated tissue and fourth-generation hDPSCs were used for the experiments.
regeneration and provided a reference for studying early angiogenesis
in the process of donor cell integration into host tissue. The hDPSCs were identified according to a previously reported
method.36 For the flow cytometry analysis of surface markers, hDPSCs
Pulpitis and pulp necrosis are common oral diseases,32 and the most were incubated with PE-conjugated anti-CD90 (12-0909-41, eBio-
traditional treatment is root canal therapy, in which the inflamed pulp science, USA; 1:20), PE-conjugated anti-CD105 (800504, BioLegend,
is removed and replaced with inorganic material, which results in a USA; 1:20), FITC-conjugated anti-CD73 (344015, BioLegend; 1:20),
nonvital tooth.33 The devitalized tooth loses nutritional supplementa- PE-conjugated anti-CD45 (304008, BioLegend; 1:20), PE-conjugated
tion to the dental hard tissue, sensitive responsiveness to hot/cold anti-CD34 (343506, BioLegend; 1:20), APC-conjugated anti-CD19
stimulation, and the ability to defend against secondary infections.34 (302212, BioLegend; 1:20), FITC-conjugated anti-CD14 (325603,
The aggregated hDPSC implantation has been verified to be effica- BioLegend; 1:20), and FITC-conjugated anti-HLA-DR (11-9956,
cious in achieving dental pulp regeneration of immature teeth whose eBioscience; 1:20) antibodies at 4 C for 30 min. The percentages of
apical foramen is not fully developed, because blood recovery and re- positively stained cells were analyzed with a CytoFLEX flow
infusion derived from host periapical tissues is relatively effective.20 cytometer (Beckman Coulter, USA) and FlowJo 10.0 software (Flow
However, for permanent teeth, in which the apical foramen has devel- Jo LLC, USA).
oped, revascularization between the intracanal implants and the sur-
rounding recipient tissue needs to be established as soon as possible to For colony formation, second-passage hDPSCs were collected, and
ensure hDPSC survival and differentiation.21 In animal models, we 1  103 cells were seeded in 10-cm culture dishes. After 10 days,
found that hDPSC-apoVs caused host-cell infiltration into the root the cells were stained with 0.1% crystalline violet (Sigma-Aldrich,
canal to reconstruct blood supply systems and pulp tissues feasibly, USA), and the number of colonies was calculated. For osteogenic
which may be a viable treatment strategy for dental pulp regeneration differentiation, hDPSCs were cultured with osteogenic culture me-
combined with stem cell application. Furthermore, it was interesting dium containing 10 mM b-glycerophosphate, 50 mg/mL ascorbic
to find de novo dentin-like tissue in the tooth scaffolds of the athymic acid, and 10 nM dexamethasone (all from Sigma-Aldrich). ALP
nude mouse models, because it suggested that apoVs may induce staining or alizarin red S (Sigma-Aldrich) staining were performed
dental-pulp endogenous regeneration, but the potential biological on day 14 or day 21, respectively. For adipogenic differentiation,
mechanisms need further exploration. hDPSCs were cultured in lipogenic medium containing 0.5 mM
isobutylmethylxanthine, 1 mM dexamethasone, 10 mM insulin,
Overall, our study provides insight into the significance of apoptosis and 0.2 mM indomethacin (all from Sigma-Aldrich) for 14 days
for pulp revascularization and tissue regeneration in an ischemic- and then stained with ORO (Sigma-Aldrich). Photographs were
hypoxic microenvironment and elucidates the mechanistic principles taken with an inverted optical microscope (Olympus, Japan).
underlying how hDPSC-apoVs regulate the angiogenesis of recipient
ECs. Our findings provide a promising approach to restore lost pulp Apoptosis inhibition
tissue based on hDPSC-apoVs ameliorating early revascularization When the cells had proliferated to 80%–90% confluence, the medium
and lay the foundation for roles played by EVs in organ repair and was supplemented with 50 mM Z-VAD(ONE)-FMK (HY-16658,
regeneration. MCE, China). One hour later, the cells were treated with 0.5 mM

microscopy images show the number of lysosomes in ECs after treatment with apoVs or Torin 1. Scale bar, 50 mm. (G) Western blot analysis shows lysosome-associated
gene (LAMP1 and CLCN7) and TFEB expressions in ECs after incubation with apoVs at concentrations of 10, 20, or 30 mg/mL. (H) Expression of TFEB in cytosolic (Cyt.) and
nuclear (Nuc.) fractions was detected by western blots after treatment with apoVs or Torin 1. (I) Representative confocal microscopy images show the locations of TFEB in
ECs after treatment with apoVs or Torin1. Scale bar, 25 mm. (J) Western blot analysis shows autophagy-associated gene (ATG7, Beclin-1, and LC3) expressions in ECs after
decreased expression of TFEB. (K) Western blot analysis shows autophagy-associated gene (ATG7, Beclin-1, and LC3) expressions in ECs after increased expression of
TFEB. After transfection with si-TFEB, the angiogenic capacity of ECs, including tube formation (L), migration (M), and proliferation (N) were detected following coculture with
apoVs (20 mg/mL). Scale bar, 100 mm; n = 3-5 per group. Data are presented as mean ± SD. Statistical analyses were performed by Student’s t test (two-tailed) for two-group
comparisons and one-way ANOVA with Tukey’s post hoc test or Welch’s ANOVA with Games-Howell post hoc test for multiple-group comparisons. *p < 0.05, **p < 0.01,
***p < 0.001; ns, p > 0.05.

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Figure 7. Autophagy is required for apoV-mediated angiogenesis in athymic nude mice model
(A) The expressions of LC3 in PBS group, apoV group, and 3-MA + apoV group were evaluated by western blots. (Bi–Di) Representative microscope images of H&E staining
show regeneration of dental-pulp-like tissue in tooth scaffolds. Scale bars, 1 mm. (Bii–Dii) Enlarged view of boxed area in (i). Scale bars, 100 mm. (Biii–Diii) Immunofluo-
rescence analysis shows the expression of blood vessel marker CD31, with positive represented by green stains. Scale bars, 50 mm; bv, blood vessels; d, dentin.

staurosporine (9953s, Cell Signaling Technology, USA) for 12 h to and 3-MA + apoV group (athymic nude mice pretreated with 3-MA +
induce apoptosis and then stained with a TUNEL assay kit (C1088, apoVs). The mice were euthanized 8 weeks after implantation, and
Beyotime Biotechnology, China). The inhibitory efficiency was deter- the tooth scaffolds were collected and fixed with 4% paraformalde-
mined by confocal microscopy (Nikon, Japan). hyde (PFA) for follow-up histological analysis.

Culture of aggregated hDPSCs In addition, we established an orthotopic model with male beagle
hDPSCs were seeded into six-well plates with 2  105 cells per well dogs (purchased from Chengdu DOSSY Experimental Animals Co.,
and cultured with a-MEM for 3 days. When the cells had reached Ltd.) by removing the dental pulp. The dogs were assigned to four
90% confluence, the medium was exchanged with a-MEM containing groups: the PBS group (treated with PBS), apoV group (treated
100 mg/mL vitamin C (Invitrogen, USA) and 10% fetal bovine serum with apoVs), Revas group (with revascularization treatment), and
(FBS), and the culture was continued for another 10 days. The normal group (normal). Preoperatively, bimaxillary CBCT was
medium was changed every 2 days. When white membrane-like performed on each beagle, and intraoperatively, gel material was
structures were observed, the cell aggregates were separated from injected into the root canal of the anterior tooth after removal
the culture plates with a cell scraper. of the pulp. After 3 months, the beagles underwent bimaxillary
CBCT, and then, the anterior teeth were extracted and fixed in 4%
Animal models PFA for 24 h at 4 C, followed by demineralization in 17% EDTA
Animal experiments were performed in accordance with the guide- (pH 7.4) for 8 weeks. The tissues were subjected to a histological
lines of the Institutional Animal Care Use Committee of the Fourth analysis.
Military Medical University and the ARRIVE guidelines.
Apoptosis analysis of the hDPSCs after implantation
Six-week-old athymic nude mice (purchased from Hunan SJA Labo- A TUNEL staining kit (C1088, Beyotime Biotechnology) was used to
ratory Animal Co., Ltd.) were procured and housed under specific- detect the apoptosis ratio of the aggregated hDPSCs after implanta-
pathogen-free conditions (24 C, 12-h:12-h light-dark cycle, and tion. Briefly, aggregated hDPSCs were removed 24, 48, or 72 h after
50% humidity) with free access to food and water. Briefly, we pre- implantation. Samples were initially frozen. The sections (10 mm
pared tooth scaffolds according to a previously reported method.36 thick) were later prepared by sequential pretreatment and then
The mice were first allocated to four groups: the hDPSC group stained with TUNEL at 37 C for 1.5 h. Cell nuclei were stained
(aggregated hDPSCs), Z-VAD group (aggregated hDPSCs pretreated with Hoechst 33342 (Sigma-Aldrich). Digital photographs were
with Z-VAD), hDPSC + apoV group (aggregated hDPSC + apoVs), taken using a laser-scanning confocal microscope (Nikon) at high
and Z-VAD + apoV group (aggregated hDPSC pretreated with magnification.
Z-VAD + apoVs). After the aggregates were filled into the tooth scaf-
folds, the tooth scaffolds were buried in the dorsum of athymic nude In addition, aggregated cells were removed 24, 48, 72, or 96 h after im-
mice under general anesthesia, with two tooth scaffolds implanted per plantation, digested to generate single-cell suspensions, and stained
mouse. To further explore the function of the apoVs, the mice were using an Annexin V/7-AAD assay kit (559763, BD Biosciences,
assigned to three groups: the PBS group (PBS), apoV group (apoVs), USA) according to the manufacturer’s instructions. The percentage

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Figure 8. Schematic diagram showing the synopsis of the findings

The apoVs released from hDPSCs was able to promote EC autophagy via transferring TUFM. Furthermore, autophagy signaling activated the angiogenesis potential of ECs.
Eventually, the accelerated revascularization promoted the efficient regeneration of dental pulp.

of apoptotic cells was analyzed using a CytoFLEX flow cytometer tants were obtained after centrifugation at 800 g for 10 min to remove
(Beckman Coulter) and FlowJo 10.0 software. the cell debris. After centrifugation at 16,000  g for 30 min at 4 C,
the precipitate was collected and washed twice with PBS. The
Histological and immunofluorescence analysis morphology of the apoVs was observed by scanning electron micro-
Tooth samples from the aforementioned animal experiments were scopy (SEM) (Thermo Fisher, USA). The size of the apoVs was deter-
collected and fixed in 4% PFA and embedded in paraffin for 24 h. mined using dynamic light scattering. The apoVs were stained with
Serial sections (5 mm thick) of the paraffin-embedded blocks were annexin V, and the expression of anti-cleaved caspase-3 (ab32042,
cut and subjected to H&E staining and Masson’s trichrome staining. Abcam, 1:1,000) was detected by western blotting.
Immunofluorescence staining was performed using standard proced-
ures. The sections were incubated with the following antibodies: hDPSC-apoVs uptake by ECs in vitro
anti-CD31 (ab28364, Abcam; 1:250), anti-DSPP (sc-73632, Santa To detect the uptake of hDPSC-apoVs by human umbilical vein ECs
Cruz Biotechnology; 1:200), and anti-DMP1 (ab103203, Abcam; in vitro, apoVs were prestained with PKH26 (Sigma-Aldrich) accord-
1:100). IgG (Abcam) was used as the negative control. The secondary ing to the manufacturer’s instructions and added to the culture me-
antibodies FITC-AffiniPure goat anti-rabbit IgG (H+L) (33107ES60, dium containing ECs. After incubation at 37 C with 5% CO2 for 12
Yeasen Biotechnology, 1:100), Cy3-AffiniPure goat anti-rabbit IgG h, the cells were washed with PBS and fixed with 4% PFA for
(H+L) (33108ES60, Yeasen Biotechnology, 1:100), and Alexa Fluor 30 min. Then, the cytoskeleton was stained with phalloidin-FITC
488 AffiniPure goat anti-mouse IgG (H+L) (Yeasen Biotechnology, (C1033, Beyotime Biotechnology), and the nuclei were stained with
1:100) were applied to the sections and incubated for 60 min. The Hoechst 33342 (Sigma-Aldrich). Fluorescent images were taken
samples were sealed with Antifade mounting medium with DAPI with a laser-scanning confocal microscope (Nikon) and analyzed
(P0131, Beyotime Biotechnology). The stained sections were viewed with ImageJ software (National Institutes of Health, USA).
with a laser-scanning confocal microscope (Nikon).
Matrigel assay
Extraction and characterization of the hDPSC-apoVs Matrigel matrix substrate gel (Corning, USA) was added to 96-well
hDPSC-apoVs were isolated from culture supernatants. First, the plates, which were then placed in a 37 C incubator for 30 min to
hDPSCs were treated with 0.5 mM staurosporine (9953s, Cell solidify the gel. ECs were seeded at a density of 1.5  104 cells per
Signaling Technology) for 12 h to induce apoptosis. Then, superna- well and incubated in serum-free medium with different doses of

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hDPSC-apoVs (0, 10, 20, or 30 mg/mL). After 4 h, tube formation was cent images were captured with a laser-scanning confocal microscope
recorded using an inverted microscope (Olympus, Japan). The tube (Nikon), and the proportion of positive cells was quantified in five
formation structure in five randomly selected fields of view in each random fields per well with ImageJ software.
well was analyzed with ImageJ software.
TFEB overexpression
Cell proliferation assay ECs were seeded at a density of 2  105 cells per well in six-well plates.
The proliferation rate of the ECs was measured by detecting MKI67/ After 24 h, the cells were transfected with a TFEB plasmid (P18466,
Ki67 (ab15580, Abcam, 1:200). A total of 1  104 ECs were seeded in Miaolingbio, China). The transfection efficiency was detected by
confocal plates and cultured with a-MEM containing 1% FBS. After western blot after 72 h.
the cells had completely adhered to the well wall, different doses of
hDPSC-apoVs (0, 10, 20, or 30 mg/mL) were added and then siRNA knockdown
incubated for 12 h. Fluorescence imaging was performed with a hDPSCs were transfected with a siRNA negative control (si-NC) and
laser-scanning confocal microscope (Nikon, Japan), and the propor- siRNA-TUFM (si-TUFM, GENECHEM) using the transfection re-
tion of positive cells in five randomly selected fields of view in each agent HitransG P (REVG005, GENECHEM) according to the manu-
plate was determined with ImageJ software. facturer’s instructions. The ECs were also transfected with siRNA
negative control (si-NC) and siRNA-TFEB (si-TFEB, RiboBio, China)
Transwell assay using a transfection kit (C10511-1, RiboBio) according to the manu-
Cell migration was quantitated using Transwell inserts with 8-mm facturer’s instructions. The transfection efficiency was measured by
pores (MCEP24H48, Corning). After 12 h of starvation, a total of western blotting after 72 h.
2  104 ECs in 200 ml of DMEM were plated in the Transwell insert,
and medium with different doses of hDPSC-apoVs (0, 10, 20 or Protein isolation and western blotting
30 mg/mL) was added to the lower Transwell chamber. After coculture Total proteins of cells and tissues were extracted using RIPA buffer
for 5 h, unmigrated ECs were removed, and migrated ECs were with added protease inhibitors. Nuclear and cytoplasmic proteins
washed with PBS, fixed with 4% PFA, stained with 1% crystal violet, were extracted with a nuclear and cytoplasmic protein extraction kit
photographed with an inverted microscope (Olympus), and counted (20126ES50, Yeasen) according to the manufacturer’s instructions.
with ImageJ software. Protein concentrations were assayed with BCA protein assay reagent
(Beyotime Biotechnology). Equal amounts of protein samples were
Proteomic analysis loaded onto SDS-PAGE gels, separated by electrophoresis and later
Protein lysates of the hDPSCs and hDPSC-apoVs were prepared. The transferred to PVDF membranes (Millipore, Germany). The membrane
peptide mixture was redissolved in a 20 mM ammonium formate so- was blocked with 5% BSA for 2 h at room temperature and then incu-
lution and analyzed by LC-MS/MS on an Orbitrap Fusion Lumos bated with primary antibody at 4 C overnight. After a wash with PBS
mass spectrometer (Thermo Fisher Scientific, USA). Tandem mass containing 0.1% Tween 20 (PBST), the membranes were incubated
spectra were processed with PEAKS Studio version X+ (Bioinformat- with secondary antibodies at room temperature for 2 h. After incuba-
ics Solutions Inc., Waterloo, Canada). Finally, 1,102 differentially ex- tion, the membranes were washed with PBST again. The blots were
pressed proteins were identified. On the basis of the Gene Ontology imaged with a Western-Light chemiluminescence detection system
(GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) data- (Tanon, China). The following primary antibodies were used: anti-
bases, we performed functional analyses of the proteins significantly cleaved caspase-3 (ab32042, Abcam; 1:1,000), anti-GAPDH (CWBIO,
upregulated in the apoVs. CW0100; 1:5,000), anti-TUBB/beta-tubulin (CWBIO, CW0098;
1:5,000), anti-HIF-1a (36169, Cell Signaling; 1:1,000), anti-VEGF
Autophagy flux assay (ab46154, Abcam; 1:1,000), anti-ANG2 (ab155106, Abcam; 1:1,000),
ECs were infected with stubRFP-sensGFP-LC3 lentivirus anti-MMP2 (ab92536, Abcam; 1:1,000), anti-Beclin-1 (3738, Cell
(GPL2001A, GENECHEM, China) for 72 h and then treated with Signaling Technology; 1:1,000), anti-LC3 (3868, Cell Signaling Technol-
apoVs for 0, 4, or 24 h. CQ (HY-17589A, MCE, China) was used to ogy; 1:1,000), anti-ATG7 (8558, Cell Signaling Technology; 1:1,000),
block the fusion of autophagosomes and lysosomes. Images were anti-TUFM (ab173300, Abcam; 1:1,000), anti-LAMP1 (ab24170, Ab-
obtained with a laser-scanning confocal microscope (Nikon) and cam; 1:1,000), anti-CLCN7 (DF3932, Affinity Biosciences; 1:1,000),
quantified with ImageJ software. anti-TFEB (4240, Cell Signaling Technology; 1:1,000), and anti-H3
(9715, Cell Signaling Technology; 1:1,000).
LysoTracker red staining
Each confocal plate was inoculated with 1  104 ECs. After the cells Statistical analysis
had completely adhered, they were cultured with fresh medium con- All the data are presented as means ± SD. Statistical and graph
taining LysoTracker Red DND-99 (40739ES50, Yeasen) for 30 min, analyses were performed with SPSS software (version 25.0). For
and then, the medium was replaced to medium containing hDPSC- two-group comparisons, significance was assessed by Student’s
apoVs, CQ (negative control; HY-17589A, MCE) or torin 1 (positive t test (two-tailed) or Student’s t test with Welch correction (two-
control; HY-13003, MCE), in three replicates of each group. Fluores- tailed). For multiple-group comparisons, significance was assessed

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Z.L., M.W., and Siying Liu contributed equally to the experimental 14. Medina, C.B., Mehrotra, P., Arandjelovic, S., Perry, J.S.A., Guo, Y., Morioka, S.,
performing, data acquisition and analysis, and manuscript drafting; Barron, B., Walk, S.F., Ghesquiere, B., Krupnick, A.S., et al. (2020). Metabolites
X.L., Y.H., and Q.Y. contributed to animal experiments; Xiaoxue released from apoptotic cells act as tissue messengers. Nature 580, 130–135.
https://doi.org/10.1038/s41586-020-2121-3.
Yang, H.G., and A.L. contributed to data analysis and interpretation;
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X.H., Xiaoshan Yang, and F.D. contributed to flow cytometry anal- emitted by dying cells. Cell Death Differ. 27, 2030–2032. https://doi.org/10.1038/
ysis; H.X., J.Z., and P.L. contributed to data interpretation; Shiyu s41418-020-0533-0.
Liu, Y.J., and K.X. contributed to the study conception and design, 16. Todorova, D., Simoncini, S., Lacroix, R., Sabatier, F., and Dignat-George, F. (2017).
data interpretation, and manuscript revision. All authors read and Extracellular Vesicles in Angiogenesis. Circ. Res. 120, 1658–1673. https://doi.org/
approved the final version of the manuscript. 10.1161/CIRCRESAHA.117.309681.
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