Small Extracellular Vesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Enhanced Proangiogenic Potential of Cardiac Fibroblasts via Angiopoietin-Like 4

Methods and Results We isolated primary CFs from Sprague-Dawley rats (1–3 days old) and treated them with lipopolysaccharide (LPS) and LPS+sEVs. RNA sequencing analysis revealed that angiopoietin-like 4 (Angptl4) was increased in the LPS+sEVs group more than in the LPS group. After inhibition of Angptl4 expression in sEVs and CFs, cell proliferation, Transwell migration, and tube formation assays were used to detect the angiogenic activity of human umbilical vein endothelial cells. β-Catenin expression in CFs was detected by western blotting. The β-catenin inhibitor ICG001 was used to examine whether β-catenin was involved in the proangiogenic potential of CFs promoted by sEVs. sEVs enhanced the proangiogenic potential of CFs under inflammatory conditions, which was associated with β-catenin signaling. The proangiogenic potential of CFs was decreased when Angptl4 was knocked down in CFs and in hucMSCs. Conclusions The sEVs regulated CFs to promote angiogenesis via Angptl4 in an inflammatory environment. This may provide a research basis for treating myocardial injury with sEVs.


Introduction
Acute myocardial injury has a high mortality rate. The repair mechanisms after myocardial injury include inflammation suppression, enhanced angiogenesis, reduced fibrosis, and remodeling. Angiogenesis and restoration of blood supply are crucial for inflammation clearance and injury repair [1]. Therefore, promoting angiogenesis is considered a good choice for repair of the myocardial injury. Cardiac fibroblasts (CFs) are major noncardiomyocytes and play an important role in remodeling of damaged tissues [2]. The proangiogenic potential of CFs has also been reported. Sar-aswati et al. isolated two different fibroblast subtypes from mouse hearts after myocardial infarction and demonstrated that FSP1+ fibroblast had a proangiogenic role [3]. The role of CFs in cardiac injury repair can shift from inflammation to angiogenesis [4,5]. It has been proven that mesenchymal stem cells (MSCs) or exosomes promote the repair of tissue injury and angiogenesis [6,7]. Li et al. showed that stem cellderived small extracellular vesicles (sEVs) promoted cardiac angiogenesis by delivering miR-486-5p, which is related to fibroblastic matrix metalloprotein 19 [8]. Exosomes derived from human endothelial progenitor cells increase proliferation and angiogenesis of CFs [9]. Our previous studies have demonstrated that exosomes derived from human umbilical cord MSCs (hucMSC-exs) had cardioprotective effects [10]. The hucMSC-exs also promote fibroblast-to-myofibroblast differentiation in the inflammatory stage and have cardioprotective effects [11]. However, whether sEVs can regulate CFs to promote angiogenesis in an inflammatory environment requires further research.
In this study, we isolated primary CFs from Sprague-Dawley (SD) rats (1-3 days old) and demonstrated that sEVs enhanced proangiogenic potential of CFs. The volcano map of differentially expressed genes (DEGs) and enrichment analysis of angiogenesis-related genes after RNA sequencing (RNA-seq) analyses of CFs showed that Angptl4 might be a noteworthy gene. The angiopoietin-like 4 (Angptl4) is a member of the angiogenin-like protein family and is a secreted glycoprotein [12]. Therefore, we investigated the proangiogenic potential of Angptl4 in CFs in an inflammatory environment.
Several studies have demonstrated that the Wnt/βcatenin signaling pathway participated in regulating angiogenesis [13]. Angptl4 is also associated with β-catenin [14]. In this study, the role of β-catenin in the activity of CFs enhanced by sEVs was also investigated.

Materials and Methods
2.1. Animals. All animals' experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Experiment Center of Jiangsu University in Zhenjiang, Jiangsu Province, China. SD rats (1-3 days old) were used in this study.

Cell
Culture. hucMSCs were isolated and cultured as described previously [15]. The umbilical cords were from the Affiliated Hospital of Jiangsu University (Jiangsu, China), and all providers gave informed consent. hucMSCs were cultured in Minimal Essential Medium Alpha (α-MEM; Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS; Biological Industries, Beit HaEmek, Israel) at 37°C in 5% CO 2 . The primary CFs were isolated from the hearts of 1-3-day-old SD rats according to an established method [16]. Human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in highglucose Dulbecco's Modified Eagle's Medium (H-DMEM; Gibco) with 10% FBS at 37°C in 5% CO 2 .
2.3. The sEV Extraction. When the hucMSCs reached 80% confluence in complete α-MEM, complete medium was replaced with α-MEM supplemented with 10% sEVsdepleted FBS. After 48 h, the supernatant was collected and centrifuged at 300 × g for 10 min, 2,000 × g for 20 min, and 10,000 × g for 30 min to remove debris. The supernatant was concentrated with 100 kDa molecular weight cut-off (MWCO) ultrafiltration centrifuge tubes (Millipore, Billerica, MA, USA). The concentrated supernatant was centrifuged at 120,000 × g for 70 min. The sedimentation at the bottom was resuspended in PBS. The mixture was centrifuged again at 120,000 × g for 70 min, and the sediment that contained the sEVs was resuspended in PBS and stored at -80°C.
2.4. Nanoparticle Tracking Analysis (NTA). The isolated sEVs were diluted with PBS. The concentration and size distribution of sEVs were evaluated by using a Malvern Panalytical nanosight nanoparticle analyzer (Malvern, UK). The data were analyzed by using ZetaView version 8.05.12 SP1 software.
2.5. Electron Microscopy. The sEVs were mixed and 20 μL was added to the sample loading copper mesh with a diameter of 2 mm, and an excess sample was removed. The copper mesh was inverted in a 2% phosphotungstic acid drop for 5 min at room temperature for negative staining. After drying, images were obtained with a transmission electron microscope (JEOL, Tokyo, Japan).

Western
Blotting. The proteins were isolated with RIPA lysis buffer containing protease and phosphatase inhibitors, PMSF, separated by 10-15% SDS-PAGE, and transferred to a PVDF membrane (Millipore, USA). Next, the PVDF membranes were blocked with 5% milk for 1 h and incubated overnight at 4°C with primary antibodies against CD9 (1 : 1,000; Cell Signaling Technology), TSG101 HUVECs/well were added to the surface of 100 μL Matrigel (Corning, NY, USA) in a 96-well plate and incubated for 6 h at 37°C. Three randomly selected fields were photographed by using an inverted microscope (Nikon). Tube formation numbers and branch points were analyzed by using ImageJ software.
2.17. Statistical Analysis. Data were given as mean ± standard deviation. Student's t-test or one-way ANOVA with the post hoc test was used to compare experimental and control groups. All data were analyzed with GraphPad Prism 8.0 software. P < 0:05 was considered to indicate a significant difference.

Results
3.1. Characterization of hucMSCs, sEVs, and CFs. To identify the hucMSCs, flow cytometry was used to detect the surface antigenic profile of the cells. CD90, CD29, and CD105 were highly expressed in hucMSCs, but CD34, CD19, and CD45 were not expressed (Figure 1(a)). The morphology of hucMSCs was spindle-shaped ( Figure 1(b)). Transmission electron microscopy (TEM) showed that the isolated vesicles had typical membrane structures (Figure 1(c)). NTA revealed that the size of sEVs was 50-160 nm (Figures 1(d) and 1(e)). To further identify the characterization of sEVs, we detected the expression of exosomal marker proteins by western blotting. CD9, TSG101, and CD81 were expressed in hucMSCs and sEVs, but calnexin was only expressed in hucMSCs (Figure 1(f)). We also measured the surface proteins of CFs by immunofluorescence and western blotting. α-SMA, vimentin, collagen I, and periostin were expressed in primary CFs, while CD31 was not (Figures 1(g) and  1(h)). These results provided sufficient support for subsequent experiments.
3.2. The sEVs Enhanced Proangiogenic Potential of CFs. To investigate whether sEVs regulated CFs to promote angiogenesis in inflammatory environment, we treated primary CFs with LPS or LPS+sEVs for 24 h and then collected the supernatants. HUVECs were cultured with the supernatants for 24 h. The supernatants of CFs pretreated with sEVs   (Figure 2(a)). Additionally, the proliferation-related protein cyclin D1 was upregulated in the LPS+sEVs group compared with the LPS group (Figures 2(b) and 2(c)). The results from wound healing and Transwell assays indicated that migration of HUVECs was increased in the LPS+sEVs group compared with the LPS group (Figures 2(d)-2(g)).
The tube formation assay revealed that tube formation numbers and branch points were enhanced in the LPS+sEVs group compared with the LPS group (Figures 2(h)-2(j)).

Angptl4
Was Increased in CFs Treated with sEVs. The sEVs enhanced proangiogenic potential of CFs. To further explore the mechanism, RNA-seq analysis of CFs was performed to find the DEGs in the LPS and LPS+sEVs groups. The volcano map of DEGs and enrichment analysis of angiogenesis-related genes showed that Angptl4 might be an important candidate gene (Figures 3(a) and 3(b)). To verify the sequencing data, qRT-PCR suggested that sEVs increased mRNA expression of Angptl4 in CF in inflammatory environment (Figure 3(c)). Additionally, the protein level of Angptl4 was also increased in the LPS+sEVs group (Figures 3(d) and 3(e)). Immunofluorescence staining showed that Angptl4 was upregulated in the LPS+sEVs group compared with the LPS group (Figures 3(f) and 3(g)).

Inhibition of Angptl4 in CFs and sEVs Resulted in
Decreased Angiogenesis. Our previous studies have demonstrated that exosomes could be taken up by CFs [11,17]. Moreover, it was shown that in Figures 3(d) and 3(f), the protein level of Angptl4 was significantly increased in the LPS+sEVs group compared with the LPS group. The expression of Angptl4 in sEVs and hucMSCs was measured. The results showed that Angptl4 was expressed in sEVs (Figure 4(a)). There are two possible mechanisms. sEVs upregulated the expression of Angptl4 in CFs, or sEVs delivered Angptl4 to CFs. Angptl4 was knocked down in hucMSCs and CFs by siRNA. Western blotting showed that siRNA3 had the best knockdown effect in hucMSCs and CFs (Figures 4(b) and 4(c)). Angptl4 was knocked down in sEVs by an Angptl4 siRNA3 (Figure 4(d)). Moreover, compared with the NC group, Angptl4 expression in CFs was significantly decreased in the LPS+si-CF+si-sEVs group (Angptl4 was knocked down in sEVs and CFs by Angptl4 siRNA3) (Figure 4(e)). Additionally, we assessed the effect of Angptl4 on angiogenesis. The results suggested that proliferation and migration of HUVECs were decreased when Angptl4 was knocked down in sEVs and CFs (Figures 4(f)-4(h)). Furthermore, as shown in Figures 4(i)-4(k), the branch points and tube formation numbers were all reduced in the LPS+si-CF+si-sEVs group. These results indicated that Angptl4 was a key molecule in regulating CFs to promote angiogenesis.

Proangiogenic Potential of CFs Enhanced by sEVs Was
Associated with β-Catenin. The Wnt/β-catenin signaling pathway is involved in angiogenesis [18]. Angptl4 is also associated with β-catenin in skin dermal fibroblasts [14]. Although the interaction between Angptl4 and β-catenin has been described in other studies, the relationship in CFs has not been studied. Moreover, the regulation of Angptl4 and β-catenin in angiogenesis is unknown in CFs. Therefore, we detected βcatenin expression in CFs by western blotting. β-Catenin was increased in the LPS+sEVs group compared with the LPS group. After inhibiting Angptl4 expression in CFs and hucMSCs, expression of β-catenin was also decreased ( Figure 5(a)). Therefore, Angptl4 regulated CFs to promote angiogenesis, possibly associated with β-catenin. To further elucidate the role of β-catenin in angiogenesis, CFs were treated with the β-catenin inhibitor ICG001. The most suitable inhibitor concentration was 10 μM (Figure 5(b)). Moreover, as shown in Figure 5(b), the proliferation of HUVECs was decreased after treatment with ICG001 (10 μM) compared with that in the LPS+sEVs group. The Transwell assay revealed that migration of HUVECs was also inhibited by ICG001 (Figures 5(c) and 5(d)). The tube formation (h-j) Tube formation ability of HUVECs was measured by the tube formation assay (h). Quantitative analysis of the total branching points (i) and tube formation numbers (j). Scale bar: 500 μm. * P < 0:05, * * P < 0:01, * * * P < 0:001, and * * * * P < 0:0001.

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Stem Cells International assay indicated that the branch points and tube formation numbers were all reduced after treatment with ICG001 ( Figures 5(e)-5(g)). These results suggested that β-catenin signaling was important for proangiogenic potential of CFs enhanced by sEVs.

Discussion
It has been demonstrated that exosomes derived from MSCs can promote angiogenesis after myocardial infarction, thereby improving heart function [8]. In addition, CFs as major cells have proangiogenic properties in the proliferation phase after myocardial infarction [19]. In our previous work, it was revealed that exosomes promoted fibroblastto-myofibroblast differentiation in the inflammation phase after myocardial infarction and exert cardioprotective effects. In this study, sEVs regulated CFs to promote angiogenesis in the inflammatory environment. Angptl4 is known to be associated with angiogenesis. Combined with RNA-seq data, we found that Angptl4 was increased in CFs after sEVs   9 Stem Cells International treatment. Further results showed that sEVs also contained Angptl4 protein. It is possible that sEVs delivered Angptl4 to CFs or sEVs upregulated the expression of Angptl4 in CFs to promote angiogenesis. After Angptl4 was knocked down in sEVs and CFs by Angptl4 siRNA3, the angiogenic potential of CFs was decreased, which indicated that sEVs enhanced the proangiogenic potential of CFs via Angptl4. During the inflammatory stage, promoting angiogenesis may have a beneficial effect on the recovery of heart function.
It was reported that β-catenin was involved in angiogenesis [20]. Our findings suggested that expression of βcatenin in CFs was decreased after inhibiting Angptl4 expression in CFs and hucMSCs. Moreover, the proangiogenic ability of CFs enhanced by sEVs was reduced by the β-catenin transcription inhibitor ICG001. It has been shown that Angptl4 was expressed in the Spemann organizer of Xenopus laevis embryos and acted as a Wnt antagonist to promote notochord formation and prevent muscle differentiation [21]. Angptl4 binds to cadherin 11, releasing membrane-bound β-catenin to accelerate wound closure [14]. However, the interaction between Angptl4 and βcatenin has not been explained in studies of myocardial injury repair. The present study demonstrated that sEVs regulated CFs to promote angiogenesis via Angptl4. However, there were complex cell populations, including cardiomyocytes, fibroblasts, endothelial cells, and inflammatory cells in the inflammatory phase after myocardial infarction. The interactions among them and proangiogenic potential have been demonstrated [22]. Cardiomyocytes, macrophages, and fibroblasts secrete proteins, sEVs, or microvesicles, mediating intercellular communication after myocardial injury. Which cell type involves increased expression level of Angptl4, promoting angiogenesis and further improving heart function, has not been explored.
In recent years, sEVs therapy has become the focus in myocardial injury repair, although there are some challenges such as efficacy and yield issues. However, the advantages of sEVs are still promising. The sEVs drugs have gradually reached the clinical development stage and have become the next generation of potential drug delivery technology [23]. In this study, we found that sEVs regulated CFs to promote angiogenesis by increasing Angptl4 expression or delivering Angptl4 in an inflammatory environment. We elucidated the role of CFs in angiogenesis early after myocardial injury, which may provide a new idea for further research on the repair mechanism of myocardial injury and therapeutic applications of sEVs.

Conclusions
Our study demonstrated that sEVs regulated CFs to promote angiogenesis via Angptl4 in an inflammatory environment. This may provide an experimental basis for research or treatment of myocardial injury with sEVs.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors confirm that they have no conflicts of interest.

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Stem Cells International provided the experimental materials. YYZ designed and supervised the study. All authors have read and approved the final manuscript.