Exosomes From LPS-stimulated hDPSCs Promote the Angiogenesis of HUVECs

Background: Angiogenesis is fundamental to biomimetic pulp regeneration. Extracellular vesicles (EVs) derived from human dental pulp stem cells (hDPSCs) from patients with periodontitis were reported to have better angiogenic capabilities. However, the underlying regulatory mechanism remains unknown. As an important component of EVs, exosomes from hDPSCs were indicated to play a crucial role in multiple regeneration processes. In this study, the inammatory factor lipopolysaccharide (LPS) was used to stimulate hDPSCs, and exosomes were extracted from these hDPSCs. The role of exosomes in the angiogenesis of Human Umbilical Vein Endothelial Cells (HUVECs) was examined, and the underlying mechanism was studied. Method: Exosomes were isolated from hDPSCs with or without LPS stimulation. The angiogenic capabilities of HUVECs were evaluated after coculture with exosomes derived from hDPSCs (hDPSC-EXOs) or exosomes derived from LPS-stimulated hDPSCs (LPS-hDPSC-EXOs). Tube formation and migration assays were conducted, and angiogenesis-related mRNA expression was detected. MicroRNA sequencing was performed to explore the microRNA prole of hDPSC-EXOs and LPS-hDPSC-EXOs. Gene Ontology (GO) analysis was used to study the functions of the predicted target genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was used to estimate the signaling pathways associated with the inammation-induced angiogenesis process. Result: The release of hDPSC-EXOs increased after stimulation with LPS. Compared to endocytosis of hDPSC-EXOs, endocytosis of LPS-hDPSC-EXOs promoted the tube formation and migration of HUVECs. The mRNA expression levels of vascular endothelial growth factor (VEGF) and kinase-insert domain-containing receptor (KDR) in the LPS-hDPSC-EXOs group were signicantly higher than those in the hDPSC-EXOs group. MicroRNA sequencing showed that 10 microRNAs were signicantly changed in LPS-hDPSC-EXOs; of these microRNAs, 7 were increased, and 3 were decreased. Pathway analysis showed that the genes targeted by differentially expressed microRNAs were involved in multiple angiogenesis-related pathways. Conclusion: This study revealed that exosomes derived from inammatory hDPSCs displayed a stronger effect on vascular regeneration. It’s the rst time to explore the role of exosomal microRNA from hDPSCs in inammation-induced angiogenesis. This nding sheds new light on the effect of inammation-stimulated hDPSCs on tissue regeneration. In the current study, we found that LPS-hDPSC-EXOs displayed a stronger effect on promoting the angiogenesis of HUVECs than hDPSC-EXOs. Our study also showed that the altered expression of certain exosomal microRNAs might be the reason for the enhanced proangiogenic ability of LPS-stimulated hDPSCs. To the best of our knowledge, this is the rst study to demonstrate the role of exosomal microRNAs from hDPSCs in the inammation-induced angiogenesis. The current study may shed light on the effect of inammation-stimulated hDPSCs on tissue regeneration.


Introduction
Stem cell-based dental pulp regeneration has been considered a novel approach for the treatment of in amed pulp tissue. However, revascularization in pulpal tissue remains the greatest challenge in biomimetic pulp regeneration. As vascular system reconstruction is a prerequisite for nutrient and oxygen transportation, angiogenesis plays a fundamental role in pulp regeneration.
hDPSCs are a type of mesenchymal stem cell (MSC) with excellent pluripotency and proliferation potential. hDPSCs can be separated conveniently and noninvasively from extracted teeth. Located in a neurovascular niche, hDPSCs have strong potential for neurogenesis and angiogenesis [1]. Many studies have shown that hDPSCs perform their proangiogenic function by guiding endothelial cells [2]. The conditioned medium, secretome, and cytokines from hDPSCs were proven to promote endothelial cell migration and tubulogenesis, and these ndings indicated the importance of the paracrine mechanism in the revascularization process [3][4][5]. In response to in ammatory stimulation, immunoregulation and regenerative events could be induced by hDPSCs. In these cases, hDPSCs show great clinical value in dental pulp repair and regeneration.
hDPSCs exhibit strong regeneration potential in controlled in ammatory microenvironments, and this potential includes strong differentiation potency and great cellular proliferation, migration, and homing abilities [6]. A similar phenomenon was observed in terms of angiogenesis. Increased blood vessel density was observed in pulpal tissues from deep caries and pulpitis [7]. In response to stimulation with lipopolysaccharides (LPS), vascular endothelial growth factor (VEGF) expression could be induced in hDPSCs via mitogen-activated protein kinase (MAPK) signaling [8]. However, the mechanism of the proangiogenic effects of in ammatory hDPSCs remains unclear.
Exosomes, a crucial element in paracrine mechanisms, are an important means of intercellular communication [9]. As a type of extracellular vesicles (EVs) with a diameter of 30-150 nm, exosomes display favorable safety and stability. Exosomes can migrate in certain directions. The complex cargo contained in exosomes can re ect the state of the parental cells [10]. All these advantages make exosomes a promising cell-free therapeutic tool for regeneration. MSC-derived exosomes display regulatory functions via mRNA, microRNA, and protein transfer [11]. It has been proven that the angiogenesis of target cells can be regulated by microRNAs from exosomes [12]. Xian et al. showed that exosomes from dental pulp cells could promote the proliferation, cytokine expression, and tube formation of human umbilical vein endothelial cells (HUVECs) via p38 MAPK signaling [5]. Interestingly, EVs secreted by in ammatory hDPSCs showed superior abilities in new vessel formation and cutaneous wound healing compared to EVs secreted by healthy teeth. Taken together, these results raised the question of whether exosomes from in ammatory hDPSCs contribute to improved angiogenic abilities [13]. In this study, we hypothesized that exosomes derived from hDPSCs from the in ammatory environment have stronger proangiogenesis effects, and these properties are mediated by speci c exosomal microRNAs.
In this study, exosomes derived from LPS-stimulated hDPSCs were isolated and characterized. The angiogenic abilities of the exosomes was studied. In addition, microRNA expression pro les of LPSstimulated hDPSC-derived exosomes were analyzed to elucidate the role of microRNAs and the underlying mechanism. To the best of our knowledge, this is the rst study to reveal that exosomal microRNAs from in ammatory hDPSCs can promote the angiogenesis of HUVECs.
Materials And Methods 1. hDPSC isolation, culture, and identi cation Third molars without periodontitis or caries from healthy human donors (aged 18-24 years) were extracted and collected at the Department of Oral and Maxillofacial Surgery, Nanfang Hospital, Guangzhou, China. This study was approved by the Ethics Committee of Nanfang Hospital, Southern Medical University. Informed consent was obtained from each patient. Pulp tissues were digested to isolate hDPSCs [14,15]. Subsequently, the hDPSCs were cultured in Dulbecco's modi ed Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (HyClone, NY, USA), in a 5% CO 2 atmosphere at 37 °C. Flow cytometry (Becton Dickinson, Tokyo, Japan) was conducted to identify stem cell surface markers. Passage 3 hDPSCs were suspended at a nal density of 5 × 10 5 cells/ml and incubated with conjugated human antibodies, including CD29-PE, CD90-PE, CD34-PE, CD45-FITC, CD44-FITC, and CD90-FICT (BD Pharmingen, Franklin Lakes, NJ) in the dark for 1 hour at 4 °C. After washing with phosphate-buffered saline (PBS; Corning, NY, USA), the cells were subjected to ow cytometric analysis.

Multilineage differentiation assay
Osteogenic and adipogenic induction were performed to determine the multilineage differentiation potential of the hDPSCs. Passage 3 hDPSCs were cultured in 6-well plates for 14 days. In the osteogenic induction group, 100 nM dexamethasone, 10 mmol/L β-glycerophosphate, and 50 mg/mL ascorbic acid (Sigma, St Louis, MO, USA) were added to the culture medium, and the mineralized nodules were stained with 2% Alizarin red S (Alizarin Red S A5533, Sigma-Aldrich). In the adipogenic differentiation group, 1 mmol/L dexamethasone, 0.05 mmol/L methyl isobutyl xanthine, 10 mg/mL insulin, and 200 mmol/L indomethacin (Sigma, St Louis, MO, USA), were added to the culture medium, and the lipid droplets were visualized by oil red O staining following a standard protocol.

cell viability assay
The hDPSCs were seeded in 96-well plates at a density of 2 × 10 3 cells/well and were stimulated with different concentrations of LPS (Sorlarbio, Beijing, China; 0, 1, 5, 10 and 50 µg/mL) for 2 days. Ten microliters of Cell counting kit-8 reagent (CCK-8; Beyotime Biotechnology, Shanghai, China) was added to each well. After 2 hours of incubation in the dark, the absorbance was measured at a wavelength of 490 nm using a microplate reader (BioTEK, Swindon, UK). Triplicate repeats were used in this assay.

Exosome-free serum preparation and exosome collection
Fetal bovine serum was diluted in DMEM to 20%. Overnight ultracentrifugation at 100,000 g was performed to eliminate the serum-derived exosomes [16]. After reaching 70% con uence, hDPSCs (passages 3 to 5) were cultured in DMEM containing 10% exosome-free bovine serum and 1% penicillinstreptomycin with or without 5 µg/mL LPS for 2 days. The culture medium was collected for exosome puri cation by programmed centrifugation. The culture medium was centrifuged at 300 × g for 10 min, and the supernatant was harvested for another centrifugation at 2,000 × g for 10 min. To remove the extracellular vesicles and apoptotic bodies, the supernatants from the previous step were collected and centrifuged at 10,000 × g for 30 min. To purify the exosomes, the supernatants were ultracentrifuged (Optima XPN-100, Beckman Coulter, USA) at 100,000 × g for 70 min. The sedimentary pellet was resuspended in phosphate-buffered saline (PBS) and then ultracentrifuged at 100,000 × g for another 70 min. The exosome pellet was resuspended in 20 µL PBS and stored at -80℃.

Exosome identi cation and BCA protein assay
The protein concentration of the exosomes was quanti ed with a micro BCA Protein Assay Kit (Thermo Fisher, USA). Transmission electron microscopy (TEM) was used to identify the exosome morphology. The exosomes were pipetted onto formvar/carbon-coated TEM grids at room temperature. After staining with 4% uranyl acetate, images of the exosomes were captured by TEM (JEM-1400 PLUS,Tokyo, JAPAN). The particle diameter was determined by Nanoparticle Tracking assay (NTA) with a Nanosight NS300 (Malvern, Worcestershire, UK). The exosomal surface markers CD9, CD63 and heat shock protein 70 (HSP70; System Biosciences, PA, USA) were examined using automated Western blotting. 6. Exosome endocytosis assay PKH67 (0.4 µL, Sigma-Aldrich, St Louis, MO) was added to 200 µL Diluent C and incubated with 20 µL exosomes for 2 min at room temperature. Then, 200 µL exosome-free FBS was added to terminate the reaction. The exosomes were washed in PBS and ultracentrifuged at 100,000 × g for 70 min. HUVECs were cultured in an endothelial growth medium-2 bullet kit (EGM-2; Lonza CC-3162, MD, USA) at 37 °C with 5% CO2. PKH67-labeled exosomes were added and incubated for 4 hours at 37 °C. The HUVECs were xed with 4% paraformaldehyde for 20 min. Antifade Mounting Medium with 4',6-diamidino-2phenylindole (DAPI; Beyotime Biotechnology, Shanghai, China) was used for nuclear staining. The images of exosome endocytosis by HUVECs were captured with an electric inverted microscope (Olympus, Tokyo, Japan).

Tube formation assay for angiogenesis
HUVECs were pretreated with exosomes derived from hDPSCs (hDPSC-EXOs) or exosomes derived from LPS-stimulated hDPSCs (LPS-hDPSC-EXOs) (100 µg/mL) for 24 hours. An equal volume of PBS was added to the control group. HUVECs were resuspended, seeded onto Matrigel (150 µL) (BD Biosciences, San Jose, CA)-precoated 48-well plates at a density of 10 5 cells/well, and incubated at 37 °C for 1 to 9 hours. Exosomes or PBS was added to each well. Images of tube formation were obtained with microscope. The indexes of tube formation were analyzed by ImageJ software.

Migration assay
A scratch wound healing assay was used to estimate the migration ability of HUVECs in response to hDPSC-EXOs or LPS-hDPSC-EXOs. HUVECs were seeded in 12-well plates at a density of 1 × 10 5 cells/well. After reaching 80% con uence, a scratch was made with a sterile pipette tip in each well. After washing with PBS, the HUVECs were exposed to fresh culture medium with hDPSC-EXOs or LPS-hDPSC-EXOs (100 µg/mL). An equal volume of PBS was added to the control group. Images of scratches were captured at 0 hours, 12 hours, and 24 hours. 9. MicroRNA sequencing A total of 3 µg RNA was extracted from each exosome sample and sent to Novogene Co., Ltd. (Beijing, China) for the construction of a small RNA library. After cluster generation, the libraries were sequenced on an Illumina HiSeq 2500 platform (Illumina, CA, USA), and 50-bp single-end reads were generated. A Pvalue of 0.05 was set as the threshold for signi cant differential expression by default. Differentially expressed microRNAs were analyzed. The microRNA target genes were predicted by two bioinformatics tools (miRanda and RNAhybrid). Gene Ontology (GO; http://geneontology.org/) enrichment analysis was

Statistical analysis
Each experiment was repeated in triplicate. All the values are presented as the mean ± SD and were analyzed in SPSS 19.0 (SPSS Inc., USA). A paired t-test was used for two-group comparisons. One-way analysis of variance (ANOVA) followed by Dunnett's T3 was used for multiple group comparisons. p < 0.05 was regarded as statistically signi cant.

Isolation and characterization of hDPSCs
The hDPSCs were extracted from healthy human third molars. The primary cultured dental pulp stem cells grew around the tissue mass (Fig. 1A). Morphological observation showed cells with broblast-like appearances (Fig. 1B). The Alizarin red staining and oil red O staining results showed that the hDPSCs could be differentiated into osteoblasts and adipocytes, respectively (Fig. 1C, D), indicating the multilineage differentiation potential of hDPSCs. The surface markers of the cells were detected by ow cytometry. The results re ected that the hDPSCs expressed high levels of the mesenchymal stem cell markers CD90 (90.5%), CD44 (99.27%), and CD29 (99.63%) and expressed minimal levels of the hematopoietic cell markers CD45 (0.16%), CD34 (0.32%), and CD105 (3.49%), indicating the mesenchymal lineage of hDPSCs (Fig. 1E-J).

Characterization of exosomes from LPS-stimulated hDPSCs.
hDPSCs were stimulated with different concentrations of LPS (0, 1, 5, 10 or 50 µg/mL). The CCK-8 assay showed the effect of LPS on hDPSC viability. No signi cant difference was observed between the 0, 1 and 5 µg/mL groups. However, compared to that in the control group, the cell viability in the 10 and 50 µg/mL groups was decreased to 70% and 62%, respectively ( Fig. 2A). The IL-6 and TNF-α expression levels were used as indicators in the in vitro in ammation model. After stimulation with LPS for 24 hours, the IL-6 and TNF-α expression levels in the hDPSCs were obviously increased in a dose-dependent manner (P < 0.05) (Fig. 2B). LPS (5 µg/ml) was used as the optimal concentration for stimulation, since this concentration could induce an in ammatory microenvironment without reducing cell viability.
Exosomes from hDPSCs were treated with or without LPS stimulation for 2 days and were harvested by programmed ultracentrifugation. TEM was used to detect the shapes of the exosomes. The extracellular vesicles from the hDPSCs presented a typical exosome shape, that is, they were round cup-shaped with a bilayer membrane (Fig. 2C). To accurately measure the different particle sizes, NTA was used. Most of the exosome diameters ranged from 30 to 150 nm, which was consistent with the standard size of exosomes (Fig. 2D). Finally, exosome-speci c markers (CD9, CD63, and HSP70) were detected by Western blotting (Fig. 2E). These results indicated that the main content of the puri ed extracellular vesicles was exosomes.
The BCA assay results showed that the hDPSCs in the LPS-induced in ammatory microenvironment produced more exosomes than those in the normal microenvironment. Furthermore, there was a positive correlation between LPS concentration and exosome volume. The exosome production from 5 µg/mL LPS-treated hDPSCs was higher than that from 1 µg/mL LPS-treated hDPSCs (Fig. 2F).

Exosomes derived from LPS-stimulated hDPSCs promoted the angiogenesis and migration of HUVECs
The hDPSC-EXOs and LPS-hDPSC-EXOs labeled with PKH-67 were taken up by HUVECs and were mainly located in the cytoplasm (Fig. 3A). This result indicated that exosomes could be a vehicle for intercellular communication.
A scratch wound healing assay was used to evaluate the effect of LPS-hDPSC-EXOs on the migration capability of HUVECs. At 12 hours, the HUVECs in the LPS-hDPSC-EXOs group exhibited signi cantly increased motility compared to the HUVECs in the hDPSC-EXOs group and control group. At 24 hours, the boundaries of the scratches in both the hDPSC-EXOs group and LPS-hDPSC-EXOs group were remarkably smaller than those in the control group (Fig. 3B). The results above showed that the HUVECs in the LPS-hDPSC-EXOs group exhibited a stronger migration ability than those in the hDPSC-EXOs group.
To investigate the different angiogenic effects of hDPSC-EXOs and LPS-hDPSC-EXOs, HUVECs were seeded on Matrigel-coated 96-well plates. After treatment with 100 µg/mL hDPSC-EXOs or LPS-hDPSC-EXOs for 1 hours and 9 hours, the number of junction points and total tube length were analyzed. At the early stage of angiogenesis (1 hours), capillary-like structures begin to form. The chains-structures could be observed in all the groups. The total tube length and junction points were higher in the LPS-hDPSC-EXOs group than those in hDPSC-EXOs group. (P < 0.05; Fig. 4A-C). At 9 hours, the late stage of angiogenesis, endothelial vessel-like networks had formed in hDPSC-EXOs and LPS-hDPSC-EXOs groups.
The LPS-hDPSC-EXOs group exhibited the greatest tube structure and had the largest quantity in junction points and total tube lenght (P < 0.05; Fig. 4D-F). In addition, the expression of proangiogenic mRNA and antiangiogenic mRNA was estimated. Compared with those in the hDPSC-EXOs stimulation group, the VEGF and KDR mRNA levels were upregulated and the THBS mRNA levels were downregulated in the LPS-hDPSC-EXOs stimulation group (P < 0.05; Fig. 4G, H). However, compared to that in the control group, Ang-1 expression was decreased in the hDPSC-EXOs stimulation group and was not signi cantly changed in the LPS-hDPSC-EXOs stimulation group (P 0.05; Fig. 4G). The results indicated that LPS-hDPSC-EXOs displayed better angiogenesis function than hDPSC-EXOs.

Pathway and GO analysis of genes targeted by differentially expressed microRNAs
The target genes of 10 differentially expressed microRNAs were predicted by 2 bioinformatics tools (miRanda and RNAhybrid). The intersection of the target gene was used for further GO and KEGG analysis.
GO analysis of the target genes showed the most signi cant biological processes, including cellular component organization, regulation of cellular communication, and cellular development process (Fig. 6A). KEGG pathway analysis showed that the targeted genes were involved in multiple important signal transductions (Fig. 6B), including the hypoxia inducible factor-1 (HIF-1) signaling pathway (Fig. 6C), Thyroid cancer related to angiogenesis, the Toll-like receptor signaling pathway (Fig. 6D), Bacterial invasion of epithelial cells related to in ammation, and Endocytosis related to exosome uptake.
Four different online microRNA databases (TargetScan, miRTarBase, miRDB, and miRWalk) were used to lter the angiogenesis-related genes targeted by the differentially expressed microRNAs. Genes that were indicated as targets by at least 2 of the databases mentioned above were included. Genes were annotated by the DAVID Bioinformatic database (https://david.ncifcrf.gov/). According to the GO term analysis, the genes that were related to the biological process of angiogenesis are shown in the mRNA-microRNA network (Fig. 7).

Discussion
Angiogenesis is a prerequisite for and hallmark of dental pulp repair and regeneration [17]. Neovascularization allows regenerative pulp tissue to perform its physiological function by providing oxygen, delivering nutrients and facilitating immune response. hDPSCs are regarded as reliable candidates for stem cell-based regeneration strategies due to their outstanding proangiogenic abilities [18]. The proangiogenic effect of hDPSCs has been proven in vivo and in vitro [19,20]. However, whether hDPSCs display different proangiogenic abilities in an in ammatory microenvironment remains largely unknown. In a previous study, increased blood vessel density was detected in dental pulp extracted from deep caries [19,20]. This result indicates that angiogenesis may also take place in response to in ammation [22,23]. In another study, the vascular network formation of HUVECs was signi cantly enhanced in a coculture system of hDPSCs and HUVECs with the addition of TNF-α [24]. In our research, when stimulated with 5 µg/ml LPS, the hDPSCs displayed stronger angiogenesis-promoting effects on HUVECs than the normal control hDPSCs. Our nding is consistent with the studies mentioned above. We hypothesize that in the early stage of in ammation, hDPSCs may play a protective role in tissue repair by reacting to in ammatory factors and then promote angiogenesis in HUVECs. Further studies are needed to demonstrate how hDPSCs respond to different types of in ammatory factors.
hDPSCs could regulate the function of HUVECs through various kinds of intercellular communication, such as paracrine and juxtacrine communication [25]. As an important component of paracrine, exosomes carry speci c biomolecules, including proteins, mRNAs, and microRNAs. It has been widely reported that exosomes play an important role in regulating multiple regeneration processes [26,27]. In a study by Xian et al., exosomes derived from hDPSCs were shown to promote the angiogenic potential of HUVECs by inhibiting the p38 MAPK signaling pathway [5]. Under in ammatory conditions, exosomes seem to have different capabilities. In another study, when cocultured with exosomes derived from LPSpretreated hDPSCs, Schwann cells showed better migration and odontoblast differentiation abilities [28]. Furthermore, EVs from periodontitis-hDPSCs exhibited a stronger effect on angiogenesis and wound healing [13]. In our study, we demonstrated that the stronger proangiogenic paracrine activity of in ammation-induced hDPSCs was mediated by exosomes. We also observed that the release of exosomes enhanced with increased LPS concentration. Our study provides strong evidence that exosomes are crucial for stem cell-based regeneration.
The cell signaling pathways by which hDPSC-EXOs regulate angiogenesis in an in ammatory environment remain unclear. Exosomal microRNAs negatively regulate the expression of their target genes by binding to the 3'UTRs of the target genes, causing translational repression [29,30]. By conducting microRNA sequencing, we found that the expression of certain microRNAs was downregulated/upregulated in the LPS-hDPSC-EXOs. In total, the expression of 10 microRNAs was signi cantly altered in response to LPS stimulation in our study, and of these microRNAs, 7 microRNAs were increased (miR-146a-5p, miR-92b-5p, miR-218-5p, miR-23b-5p, miR-2110, miR-27a-5p, and miR-200b-3p) and 3 microRNAs were decreased (miR-223-3p, miR-1246 and miR-494-3p). Among microRNAs, 5 microRNAs have been proven to play important roles in in ammation and HUVEC function and angiogenesis. We assume that the differentially expressed exosomal microRNAs might be the reason why in ammation-stimulated hDPSCs display a stronger revascularization role.
MiR-223-3p has been con rmed to regulate the function of various systems, including the cardiovascular system and immune system [31]. In head and neck squamous cell carcinoma (HNSCC) tissues, miR-223-3p expression was negatively correlated with CD31 expression, indicating its antiangiogenic properties [32]. The mRNA and protein expression of VEGF was signi cantly increased in breast cancer cells in which miR-223-3p was inhibited in vitro [33]. Furthermore, miR-223 was deregulated in several types of in ammatory diseases, such as sepsis, type 2 diabetes, and rheumatoid arthritis [34]. Taken together, these ndings suggest that miR-223-3p is a strong candidate for the mechanism by which LPS-hDPSC-EXO-derived microRNA promotes the angiogenesis of HUVECs. We also found some other interesting observation by reviewing articles. In the study by Li et al., the expression of miR-146a was induced by LPS treatment. Angiogenesis was inhibited by miR-146a knockdown via TGF-β1 signaling pathway activation [35]. In another study, the expression level of miR-218-5p in glomerular mesangial cells (GMCs) was upregulated by LPS stimulation [36]. MiR-218-5p knockdown promoted the apoptosis of HUVECs by activating HMGB1 [37]. MiR-200b-3p was reported to affect HUVEC functions by directly regulating a variety of proangiogenic genes (e.g., VEGFA) and anti-angiogenic target genes (e.g., KLF2) [38]. A previous study revealed that miR-1246 inhibited angiogenesis by repressing NF-κB signaling [39]. Further studies are required to demonstrate how certain LPS-hDPSC-EXOs microRNAs promote the angiogenesis of HUVECs.

Conclusion
In the current study, we found that LPS-hDPSC-EXOs displayed a stronger effect on promoting the angiogenesis of HUVECs than hDPSC-EXOs. Our study also showed that the altered expression of certain exosomal microRNAs might be the reason for the enhanced proangiogenic ability of LPS-stimulated hDPSCs. To the best of our knowledge, this is the rst study to demonstrate the role of exosomal microRNAs from hDPSCs in the in ammation-induced angiogenesis. The current study may shed light on the effect of in ammation-stimulated hDPSCs on tissue regeneration.