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. Author manuscript; available in PMC: 2020 Apr 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2019 Apr;39(4):765–773. doi: 10.1161/ATVBAHA.118.311919

DKK3 Trans-differentiates Fibroblasts into Functional Endothelial Cells

Ting Chen 1,#, Eirini Karamariti 2,#, Xuechong Hong 2,#, Jiacheng Deng 2, Yutao Wu 3, Wenduo Gu 4, Russell Simpson 4, Mei Mei Wong 4, Baoqi Yu 5, Yanhua Hu 6, Aijuan Qu 7, Qingbo Xu 8, Li Zhang 9
PMCID: PMC6445366  EMSID: EMS81859  PMID: 30816803

Abstract

Objective

To determine the role of a cytokine-like protein Dikkopf-3 (DKK3) in directly trans-differentiating fibroblasts into ECs and the underlying mechanisms.

Approach and Results

DKK3 overexpression in human fibroblasts under defined conditions for 4 days led to a notable change in cell morphology and progenitor gene expression. It was revealed that these cells went through mesenchymal-to-epithelial transition and subsequently expressed KDR at high levels. Further culture in EC defined media led to differentiation of these progenitors into functional ECs capable of angiogenesis both in vitro and in vivo, which was regulated by the VEGF/miR-125a-5p/Stat3 axis. More importantly, fibroblast-derived ECs showed the ability to form a patent endothelium-like monolayer in tissue-engineered vascular grafts ex vivo.

Conclusions

These data demonstrate that DKK3 is capable of directly differentiating human fibroblasts to functional ECs under defined media and provides a novel potential strategy for endothelial regeneration.

Keywords: DKK3, trans-differentiation, endothelial cell, angiogenesis, vascular tissue engineering

Subject terms: Vascular Biology, Cellular Reprogramming, Angiogenesis

Introduction

Dickkopf-3 (DKK3), a secreted glycoprotein, is a member of the dickkopf family of Wnt inhibitors1. It has been shown that DKK3 is involved in the differentiation of partially induced pluripotent2 and embryonic stem cells3 to smooth muscle cells (SMCs)4. DKK3 was also identified to stimulate differentiation of Sca1+ vascular progenitor cells and modify the phenotype of atherosclerotic plaque5. Recently we have shown that DKK3 has a protective role in the pathogenesis of atherosclerosis via regulation of endothelial regeneration7. An attempt to clarify the mechanism involved in the regulation of angiogenesis by DKK3 indicates a correlation between this protein and vascular endothelial growth factor (VEGF)6, 7. Meanwhile the high expression of DKK3 in tumour vascularization suggests a function for DKK3 as an endothelial cell (EC) differentiation factor8. Taking these findings under consideration, it is postulated that DKK3 is a pro-angiogenic protein in both neovascularization and endothelial regeneration of large vessels.

ECs are key components of the vessel wall with immense potential in regenerative medicine and particularly cellular therapeutic strategies aiming to enhance tissue engineered constructs or rejuvenate ischemic tissues via neovascularization. Although several approaches have succeeded in generating functional ECs from various sources such as embryonic stem cells9–11, induced pluripotent stem cells12–14, progenitor cells15, 16 or directly from terminally differentiated cell lines via combination of factors2, 17, the complexity of the protocols, the low efficiencies as well as the danger of contamination due to long term cultures and cultures containing feeder layers constitute obstacles in massively and safely generating ECs for clinical applications.

In this study, we presented a fast and straightforward protocol of generating functional ECs from human fibroblasts by overexpressing DKK3, under defined culture conditions. In addition, we explored possible mechanisms and revealed the involvement of mesenchymal-to-epithelial transition (MET) and the miR-125a-5p/Stat3 axis in regulating fibroblast transdifferentiation into ECs.

Materials and Methods

The authors declare that all supporting data are available within the article (and in the online-only Data Supplement).

Materials

Chemicals, reagents, and antibodies are listed in the online-only Data Supplement.

Adenoviral Amplification, Titration and Infection

A human DKK3-HA Adenovirus (cat. No. 085672A) and a null adenovirus (cat. No. 000048A) were purchased from Applied Biological Materials and amplified according to the protocol provided by the company.

Endothelial Cell Differentiation

After 4 days of adenoviral transduction, null and human DKK3 overexpressing cells were changed to complete EGM™-2™ (EGM™ Endothelial Growth Medium (CC-3024) & EGM™ BulletKit™ (CC- 3124)) supplemented with 10ng/ml VEGF (Perpoteh, cat. No. 100-20B).

In Vitro Tube Formation Assay

Chambers of an 8 well chamber slide were coated with 50μl of Matrigel® (Corning, cat. No. 356234) at 37 °C for 30min-1h. 104 control or iECs were then seeded on top of the substrate and maintained in complete EGM-2-VEGF for 6-8h. Rearrangement of the cells and formation of capillary like structures were then observed and images were captured with an Axioplan 2 imaging microscope.

In Vivo Angiogenesis Assay

Control cells or iECs were mixed with 50 µl of Matrigel and injected subcutaneously into the back or flank of NOD.CB17-Prkdcscid/NcrCrl mice (purchased from The Jackson Laboratory, USA). Six injections were conducted for each group. 10 days later, the mice were sacrificed and the plugs were harvested, frozen in liquid nitrogen and then cryosectioned. Samples were then fixed with 4% paraformaldehyde in PBS at 4°C overnight after which HE staining was applied. Images were assessed with the Axioplan 2 imaging microscope, captured with an AxioCam camera and processed with Photoshop software (Adobe) if required.

Dual Seeding Vascular Graft Using ex vivo Bioreactor System

The thoracic aorta was excised from the mouse (male C57Bl/6 mice were purchased from Charles River, Margate, Kent, UK) and immediately flushed with saline solution containing 100U heparin to prevent the formation of blood clots. Peri-aortic connective tissue was gently removed with forceps. The vessel was harvested and cryosection was prepared for further analysis.

shRNA Lentiviral Particle Transduction

Lentiviral particles were generated using shRNA E-Cadherin (CDH1 MISSION shRNA, SHCLNG_NM_004360, Sigma Aldrich) according to the protocol previously described2. Briefly, the shRNA Non-Targeting (NT) vector was used as negative control. HEK 293T cells were tranfected with the lentiviral vector and the packaging plasmids, pCMV-dR8.2 and pCMV-VSV-G (both from Addgene) using FuGENE HD (Promega). The supernatant containing the lentiviral particles was collected and filtered 48 hours after transfection. For lentiviral transduction, cells were seeded overnight and the following day the cells were incubated with shRNA or NT control in complete medium supplemented with 10µg/ml of Polybrene for 16 hours. Subsequently fresh medium was added to the cells and the plates were harvested at the stated time points after transduction.

Statistical Analysis

The data were analyzed using Graph Pad Prism Software. Data were analyzed by two-tailed Student’s t test or analysis of variance (ANOVA) followed by multiple comparisons with Bonferroni’s method when the data are passed for the normality and equal variance tests. Data were presented as the mean and standard error of the mean (SEM). A value of P<0.05 was considered significant.

Results

DKK3 Induces a Vascular Progenitor State in Fibroblasts through MET

Human embryonic lung fibroblasts were infected with a null or DKK3 adenovirus and maintained in α-MEM containing 10% Knockout Serum Replacement (KOSR). DKK3 expression reached a higher level 2 day after infection and returned to baseline 7 days (Figure IA and IB in the online-only Data Supplement). After maintaining the cells in these conditions, it was revealed that 4 days of DKK3 overexpression were sufficient to dramatically alter the cell morphology from an elongated and fiber-like phenotype to a smaller, rounder and occasionally more spindle-like shape (Figure 1A). To elucidate the change in morphology, the expression levels of SMC and EC markers were initially investigated. It was found that most of the important SMC markers were significantly downregulated whereas the EC markers were not significantly changed (Figure IC and ID in the online-only Data Supplement).

Figure 1.

Figure 1

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Transient overexpression of DKK3 in fibroblast induced the upregulation of vascular progenitor markers through MET. (A) Human lung embryonic fibroblasts were seeded on gelatin coated flasks, infected with null or Ad-DKK3 adenovirus and maintained in α-MEM-10% KOSR medium. Change in morphology of fibroblasts after 4 days of DKK3 adenoviral overexpression. (B) qPCR showed the upregulation of KDR, CD133 and CD14 at the mRNA and (C) protein levels after 4 days of DKK3 overexpression. (D) Representative images of Immunofluorescent staining demonstrated the increased expression of KDR and CD133 in Ad-DKK3 transfected fibroblasts compared to cells transfected with null adenovirus. (E) qPCR revealed the upregulation of the epithelial marker E-Cadherin and (F) suppression of mesenchymal markers after 4 days of DKK3 overexpression. (G) The upregulation of epithelial marker E-Cadherin and downregulation of mesenchymal markers Vimentin and Slug were confirmed at the protein level. (H) DKK3 overexpressing fibroblasts were infected with null or shE-Cadherin. Protein analysis revealed a negative effect of vascular progenitor marker expression. (I) Matrigel assay. The cells were infected with vector (Null) or Ad-DKK3 adenovirus, mixed with matrigel and injected subcutaneously into SCID mice. The plugs were harvested a week after implantation, sectioned, stained for E-Cadherin (E-Cad) or Vimentin (Vim). Quantitative data are means±SEM, n=5. *Significant difference from the control, p<0.05. Ctl-, negative control stained with normal IgG.

To clarify the direction of the transformation, gene expression of the germ layers ectoderm and mesoderm was further investigated. QPCR analysis revealed that the change in the morphology of the cells was not a reflection of a transformation of the cells to any of the aforementioned layer (Figure IE and IF in the online-only Data Supplement). Furthermore, an investigation of more specific cell lineages such as cardiomyocytes and pericytes showed that the cells did not adopt a specific cardiovascular phenotype (Figure IG and II in the online-only Data Supplement).

To further characterize the newly generated cells and identify possible multipotent or progenitor characteristics, qPCR and FACS analysis were performed for both mesenchymal and vascular progenitor markers. Interestingly, it was revealed that while there was slightly downregulation in the expression of mesenchymal markers at both the RNA (Figures IG in the online-only Data Supplement) and protein (Figure IIA, IIB in the online-only Data Supplement) levels such as CD90 and CD105, the levels of some progenitor cell markers such as KDR, CD133, CD14, c-Kit, CD34 were significantly increased (Figure 1B and 1C, Figure IIC, IID in the online-only Data Supplement). Immunofluorescence staining also confirmed that some progenitor markers (KDR, CD133, c-Kit, CD34) are highly expressed in fibroblasts overexpressing DKK3 for 4 days (Figure 1D, Figure IIE in the online-only Data Supplement). Particularly, phalloidin staining nicely demonstrates the morphological changes of fibroblasts and confirms the transformation of these cells (Figure IIE in the online-only Data Supplement).

The participation of MET in modulating cellular identity has been shown in several studies18–20. Notably, qPCR analysis showed that the expression of the epithelial marker E-cadherin was induced (Figure 1E) in parallel with the suppression of the mesenchymal markers Fibroblast-specific protein 1 (FSP-1), Vimentin, Slug and Fibronectin after 4 days of DKK3 overexpression (Figure 1F and 1G). These results suggested that the generation of a vascular progenitor state occurs in parallel with a Mesenchymal-to-Epithelial Transition (MET) especially the suppression of mesenchymal signature genes. Silencing of E-Cadherin during reprogramming reduced expression of vascular progenitor markers such as CD14 compared with Ad-DKK3 group, while the expression of mesenchymal gene marker FSP-1 was induced. (Figure 1H). In vivo matrigel assay confirmed upregulation of E-Cadherin expression and downregulation of vimentin induction in Ad-DKK3 overexpressed cells (Figure 1I). Collectively, our results suggested that MET is indispensable during the transformation of fibroblasts to progenitor cells.

DKK3 Mediates Cell Differentiation into iECs via Activation of KDR Under Defined Condition

The significant induction of KDR during DKK3-induced vascular progenitor state suggested that DKK3 could regulate KDR expression through gene transcription. Luciferase assays indicated that the promoter region of KDR was activated upon DKK3 overexpression in different time points (Figure 2A and 2B). Immunofluorescence staining showed that KDR accumulated within cells with DKK3 overexpression (Figure 2C). Results also showed that the transcription repressor TFII-1 that has been associated with KDR promoter activation was downregulated after DKK3 overexpression (Figure IIIA and IIIB in the online-only Data Supplement).

Figure 2.

Figure 2

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Activation of KDR promoter by DKK3 overexpression leads to differentiation of fibroblasts to iECs under defined condition. The activation of three different portions of the KDR promoter sequence (-225 to + 268, -570 to +268, -780 to +268) by DKK3 overexpression was confirmed by Luciferase Assay at (A) 48 and (B) 72 hours. (C) Immunofluorescent staining revealed KDR nuclear accumulation (D) DKK3 overexpressing fibroblasts were further cultured in endothelial medium supplemented with VEGF for an additional 6 days which further altered their the morphology. (E) qPCR of the differentiated cells for endothelial specific markers such as CD31 and CD144. (F) Indirect immunofluorescent staining confirming upregulation of endothelial specific markers at the protein level while exhibiting characteristic staining of endothelial cells with nuclear staining (DAPI). (G) qPCR of the differentiated cells (iEC) and human umbilical cord endothelial cells (HUVECs) for endothelial specific markers. Graphic data are mean ±SEM of three independent experiments with duplicates; *P<0.05, **P<0.01.

The expression of a range of progenitor markers was an indication that the cells had entered a stage of plasticity that made them amenable to differentiation towards a specific cell type upon the right stimulus. We therefore changed the culture medium to complete endothelial EGM-2™ supplemented with VEGF for an additional 6 days. Initially, a dramatic change in cell shape was observed, where cells adopted a typical cobble stone morphology, characteristic of human endothelial cells in culture (Figure 2D). Following this observation, the expression of specific EC markers such as CD31 and CD144 were investigated at both the mRNA (Figure 2E) and protein levels (Figure IVA to IVD in the online-only Data Supplement) and confirmed by immunofluorescent staining (Figure 2F), which is comparable to those expressed in human umbilical cord endothelial cells (Figure 2G). Furthermore, the expression of angiogenic markers of endothelial cells such as ANGPT1, ANGPT2 and Tie2 were investigated. It was found that the expression of these markers was significantly upregulated in parallel with important endothelial markers such as KDR at the mRNA and protein levels (Figure IVE and IVF in the online-only Data Supplement).

Finally, to confirm the angiogenic potential of these endothelial like cells, a human angiogenic proteome profiler kit was utilized to detect 55 human angiogenesis related proteins. Analysis of the microdot plots by densitometry showed that a greater number of proteins related to angiogenesis were upregulated in the endothelial like differentiated cells as opposed to the control cells (Figure V and Supplemental Table II in the online-only Data Supplement). Although a general trend of upregulation was observed throughout the entire array, proteins such as angiogenin, amphiregulin, Epidermal growth factor (EGF), Epidermal growth-VEGF (EG-VEGF), endothelin-1 Interleukin-8 (IL-8) and Tissue inhibitors of metalloproteinase 4 (TIMP-4) presented with the most prominent upregulations as shown in the boxed areas of the microdot plot. We therefore concluded that the cells had successfully differentiated to endothelial like cells and henceforth called them induced-ECs (iECs; Figure 4E and 4F), which was comparable with human umbilical cord endothelial cells (Figure 4G).

Figure 4.

Figure 4

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Functional analysis of iECs. (A) iECs were seeded on matrigel coated 6 well chamber slides and maintained in EGM™-2™-VEGF medium for 6-8 hours. Images captured with phase contrast microscopy revealed the formation of endothelial cell networks in iECs (B) Quantification of network structures formed by control and iECs on a matrigel substrate (C, D) Cells were incubated with an Alexa Fluor® 594 Conjugated LDL antibody and images were captured using fluorescent microscopy revealing an increased LDL uptake from iECs. (E) HE staining of sections obtained from the areas of injection after 10 days revealed the generation of microvascular tubes in the areas of iEC injection. (F) Immunofluorescent staining for human CD144 (green) of frozen sections obtained from the same areas demonstrates that the labelled cells (red) forming the microvascular tubes are also positive for the human endothelial marker CD144. (G) Decellularized mouse aorta graft double seeded with DKK3-transformed-ECs and SMCs showed vessel-like structures (red: CD31, green: α-SMA) after being maintained in ex vivo bioreactor setting for 5 days and was confirmed by human specific CD31, CD144 immunostaining (H), (mean ±SEM, n=5 or more; *P<0.05).

miR-125a-5p/Stat3 Axis is Implicated in Endothelial Differentiation

Several studies have demonstrated that miR-125a-5p regulates angiogenesis by directly binding to the 3'-untranslated region (3’-UTR) of Stat3 in endothelial cells21, 22. We therefore postulated that the miR-125a-5p/Stat3 signaling pathway may be involved in the DKK3 induced iEC differentiation under defined condition. Congruently, our data revealed a consistent downregulation of miR-125a-5p as well as its precursor transcript expression (Figure VIA and VIB in the online-only Data Supplement) and upregulation of Stat3 (Figure VIC in the online-only Data Supplement) during the DKK3 induced EC differentiation.

To further investigate the involvement of miR-125a-5p in endothelial cell differentiation, we performed loss-of-function and gain-of-function experiments. Fibroblasts were overexpressed with DKK3 for 4 days and then differentiated toward ECs for an additional 6 days. At the 3rd day of differentiation, cells were transfected with either miR-125a-5p mimics or inhibitor and were harvested 48-72 hours later. Overexpression of miR-125a-5p mimics reduced the expression of EC markers such as CD144 and CD31 at both mRNA levels and protein level whereas transfection with a miR-125a-5p inhibitor enhanced the expression of the aforementioned markers respectively (Figure 3A to 3C, Figure VID and VIE in the online-only Data Supplement). Importantly, cells revealed a decreased capacity to form vascular tubes in the presence of miR-125a-5p mimics. In contrary, iEC with miR-125a-5p inhibitor displayed an increase capacity to form vascular tubes in vitro (Figure 3D to 3F).

Figure 3.

Figure 3

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miR-125a-5p/Stat3 axis is implicated in DKK3 transformed iEC differentiation. After 4 days of reprogramming, DKK3 transformed vascular progenitor cells were forced to differentiation toward EC for another 6 days, and then efficiently transfect with either miR-125a-5p mimics and inhibitor at day 3, cells were harvest on day 6. (A) miR-125a-5p mimics decreased the EC specific marker expression and miR-125a-5p inhibitor (B) increased those EC marker expression, both in mRNA and protein level (C). On the day 6, the cells have been subjected to Matrigel plugs assay in vitro, miR-125a-5p reduced the tube formation comparison to the control, where more defined vascular structures were observed in miR-125a-5p inhibition (D and quantification in E, F scale bar =50 um). Meanwhile, miR-125a-5p overexpression suppressed the expression of Stat3 in mRNA level (G-H), the result of WB is shown in (I). (J-K) DKK3 transformed ECs were co-transfected with miR-125a-5p mimics, inhibitor, relative control and Stat3 3’UTR report plasmid, luciferase assay demonstrating the 3 ’UTR of Stat3 is a direct target gene of miR-125a-5p. (L-M) Stat3 inhibition recapitulates the effects of miR-125a-5p overexpression on EC specific gene expression. (N) the effect of the miR-125a-5p inhibitor in EC markers was ablated after Stat3 suppression. All graphic data are means ±SEM from three independent experiments with duplicate samples. *Significant difference from the control, p<0.05. Abbreviation: mim ctl: mimics control, inhib ctl: inhibitor control, miR-125 mim: miR-125a-5p mimics, miR-125 inhib: miR-125a-5p inhibitor.

Further experiments demonstrated that miR-125a-5p mimics suppressed the expression of Stat3 while miR-125a-5p inhibitor induced its expression (Figure 3G to 3I). Additionally, luciferase assays revealed that the 3’UTR of Stat3 is a direct target of miR-125a-5p (Figure 3J and 3K). Interestingly, silencing of Stat3, either by siRNA or a pharmacological inhibitor, led to a robust reduction of EC specific markers as well as the in vitro tube formation during the DKK3-induced EC differentiation (Figure 3L, 3M, Figure VIIA to VIIC in the online-only Data Supplement). We also found that Stat3 silencing led to a decrease of the gene promoter activity of CD144 and KDR (Figure VIID and VIIE in the online-only Data Supplement). Furthermore, upon siRNA-mediated knockdown of the Stat3, the regulatory effect of miR-125a-5p inhibition was ablated (Figure 3N). Collectively, these data confirm that miR-125a-5p can modulate phenotypic switching and cell fate during DKK3 induced differentiation to an endothelial lineage by targeting Stat3.

iECs Exhibit Endothelial Functions

After confirming the successful differentiation from fibroblasts to iECs, assessing the functionality of iECs was of vital importance. Initially, cells were seeded on a matrigel substrate to determine their angiogenic potential in vitro. Indeed, iECs formed cell networks after 6-8 h of seeding whereas the control cells never organized into such structures (Figures 4A and 4B). Furthermore, as shown in Figure 4C and 4D, iECs were capable of uptaking ac-LDL which is the typical function of endothelial cells. Finally, null-ECs or iECs were labeled with Qtracker® (red) and subcutaneously injected into mice in the form of a matrigel plaque assay. It was revealed that iECs exhibited the ability to form microvascular tubular structures in vivo in 10 days as demonstrated by hematoxylin-eosin (HE) staining (Figure 4E) and immunofluorescent staining for CD144 (green) (Figure 4F).

An ex vivo circulation bioreactor system developed in our lab has been used to seed cells to a decellularised mouse aortic graft to construct native vessel-comparable vascular grafts3. Following seeding iEC into the decellularized graft and culturing the graft in the bioreactor circulation system for 5 days, the graft was harvested, sectioned and stained for the endothelial marker CD31 and CD144 with immunofluorescence. The reconstructed vascular graft displayed a vascular like structure with the most inner endothelium-like layer formed by DKK3 transformed iECs that were surrounded by multiple SMC layers (Figure 4G, H). Interestingly, the ability of angiogenesis in matrigel plug was significant influenced by treatment of the cells with miR-15-mimics (Figure VIII in the online-only Data Supplement). Cell transfection with miR-125a-5p mimics resulted decreaded Stat3 expression correlated with decreased angiogenesis in vivo. Hence, we concluded that the iECs generated by fibroblasts via DKK3 adenoviral induction under defined culture conditions were functional ECs.

Discussion

It is of great significance to obtain human endothelial cells of high quality in order to enhance angiogenesis in ischemic tissues and endothelial repair in large vessels and to generate functional vascular conduits. Although it has previously been shown that functional ECs can be derived from fibroblasts using reprogramming methods2 of the correct combination of transcription factors as well as the appropriate culture conditions17, 23 the simplicity of our protocol introduces for the first time a fast, one factor, straightforward trans-differentiation method to obtain functional ECs from human fibroblasts in the span of 10 days with no elaborate genetic manipulations. While DKK3 has previously been associated with SMCs differentiation 3, this study is further evidence to the notion that returning to an “unstable”, multipotent state is possible via different routes so long as the right circumstances have been generated for the cell to react to the appropriate stimulus.

Interestingly, we found that during the 4 days of reprogramming, fibroblasts gradually lost their morphology as well as their mesenchymal gene marker expression and simultaneously increased the expression of the cell-to-cell adhesion marker, E-cadherin, which is a crucial indicator of MET19. MET is a key process involved in the manipulation of cell fate plasticity20, 24. Furthermore, knockdown of E-cadherin disrupted vascular progenitor marker induction. Successful interactions between neighboring cells mediated by E-cadherin may be a requisite event for the further establishment of vascular progenitor and endothelial junction proteins. There is no change in gene expression of pluripotency marker Oct-4, Sox2, Klf4 and c-myc in the infected cells (data not shown). In addition, high KDR expression was detected in our cell model. KDR, the receptor for VEGF, is the earliest marker of the endothelial lineage to be expressed during development25. Activation of KDR in our system suggests that these cells may be responsive to VEGF stimulation and thus capable of differentiation to ECs. We therefore changed the medium to include recombinant VEGF. Our results indicated that DKK3 can successfully differentiate fibroblasts to ECs under defined condition. Importantly, these iECs displayed good attachment, typical endothelial structure and functional properties, providing an excellent source of cells for the use in regenerative therapy.

To shed light onto the mechanism by which DKK3 induces iECs differentiation, we focus on the miR-125a-5p/Stat3 axis pathway, Stat3 is well-characterized as aspect of mediated angiogenic regulation and required for endothelial differentiation in stem cells26, miR-125a-5p can regulate angiogenesis22 and has been verified as a tumor suppressor by targeting Stat3. In this study, we provide compelling evidence that Stat3 is the directly target of miR-125a-5p and mediated the DKK3 transformed iEC differentiation process by the following: 1) decrease the gene expression of endothelia specific marker such as CD144, CD31 by Stat3 suppression and miR-125a-5p overexpression; 2) reduction of Stat3 expression and its reporter activity by miR-125a-5p mimics; 3) the effect of miR-125a-5p on iEC differentiation can be abolished after Stat3 depletion. Above evidence demonstrated the novel mechanistic roles involved the miR-125a-5p/Stat3 signaling pathway during the fibroblast transdifferentiation to endothelial lineage (Figure IX in the online-only Data Supplement).

Therefore, we believe that DKK3 overexpression under the defined culture conditions can induce a progenitor like state to the cells that transforms them into a cell type amenable to appropriately respond to potent endothelial signals, bringing one factor trans-differentiation for personalized therapy within our grasp. Moreover, our method provides the ability to differentiate into ECs through defined media by “skipping pluripotency” then prevents the risk of tumor formation. This study also has implications since generation of a large population of functional ECs can fundamentally change personalized vascular therapy.

Supplementary Material

Graphic Abstract
Supplemental Material

Highlights.

*Overexpression of a cytokine-like protein DKK3 in fibroblasts led to progenitor phenotype.

*The progenitor phenotype of cells can differentiate into functional endothelial cells.

*The mechanism of cell transdifferentiation involves in mesenchymal-to-epithelial transition and VEGF-miRNA-Stat3 pathways, respectively.

Sources of Funding

This study was supported by grants from the British Heart Foundation (RG/14/6/31144) and the National Natural Science Foundation of China (81220108004, 81770435, 81570249, 91339102, 91639302 and 91539103) and the Zhejiang Provincial Natural Science Foundation (LD18H020001). T. C. was supported by a Zheng Shu Medical Elite Scholarship.

Non-standard Abbreviations and Acronyms

DKK3

Dickkopf 3

KOSR

Knockout serum replacement

ECs

Endothelial cells

SMCs

Smooth muscle cells

MET

Mesenchymal-to-epithelial transition

MiR

MicroRNA

Stat3

Signal transducer and activator of transcription factor 3

VEGF

Vascular endothelial growth factor

KDR

Vascular endothelial growth factor receptor 2

3’-UTR

3’-Untranslated region

FSP-1

Fibroblast-specific protein 1

EGF

Epidermal growth factor

IL-8

Interleukin 8

TIMP-8

Tissue inhibitors of metalloproteinase 8

EG-VEGF

Endocrine gland-derived vascular endothelial growth factor

Footnotes

Disclosures

None.

Contributor Information

Ting Chen, Department of Cardiology, the First Affiliated Hospital, School of Medicine, Zhejiang University, China.

Yutao Wu, Department of Cardiology, the First Affiliated Hospital, School of Medicine, Zhejiang University, China.

Baoqi Yu, Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital Medical University, Beijing, China.

Yanhua Hu, School of Cardiovascular Medicine and Sciences, King’s College London BHF Centre, London, UK.

Aijuan Qu, Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital Medical University, Beijing, China.

Qingbo Xu, School of Cardiovascular Medicine and Sciences, King’s College London BHF Centre, London, UK.

Li Zhang, Department of Cardiology, the First Affiliated Hospital, School of Medicine, Zhejiang University, China.

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