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Semaphorin-4D signaling in recruiting dental stem cells for vascular stabilization

Stem Cell Research & Therapy volume 16, Article number: 25 (2025) Cite this article

Abstract

Background

Achieving a stable vasculature is crucial for tissue regeneration. Endothelial cells initiate vascular morphogenesis, followed by mural cells that stabilize new vessels. This study investigated the in vivo effects of Sema4D-Plexin-B1 signaling on stem cells from human exfoliated deciduous teeth (SHED)-supported angiogenesis, focusing on its mechanism in PDGF-BB secretion. We also explored macrophages as an endogenous source of Sema4D for vascular stabilization.

Methods

The in vivo Matrigel plug angiogenesis assay was conducted to examine the impact of Sema4D on vessel formation and stabilization supported by SHED. Knockdown of Plexin-B1 in human umbilical vein endothelial cells (HUVECs) and PDGFR-β inhibitors were utilized to explore the fundamental regulatory mechanisms. Furthermore, the m6A methylation levels of total RNA and the expression of Methyltransferase-like 3 (METTL3) were assessed under conditions of Sema4D treatment in vitro. An ELISA was employed to measure the levels of Sema4D in the supernatants derived from THP-1 cell-mediated macrophages. Additionally, a three-dimensional vasculature-on-a-chip microfluidic device was used to investigate the role of M2c macrophage-derived Sema4D in the stabilization of vascular structures.

Results

Sema4D induced the formation of a greater number of perfused vessels by HUVECs and enhanced the coverage of these vessels by SM22α-positive SHED (SM22α+SHED). Conversely, the knockdown of the Plexin-B1 receptor in HUVECs or inhibition of PDGFR-β reversed the Sema4D-induced vascular stabilization, thereby confirming the regulatory role of the Plexin-B1/PDGF-BB axis in the recruitment of mural cells mediated by Sema4D. Mechanistically, Sema4D was found to upregulate the expression of methyltransferases, specifically METTL3, and to elevate the level of m6A modification in HUVECs. This modification was determined to be critical for enhancing PDGF-BB secretion, suggesting that Sema4D activates an epigenetic regulatory mechanism. Additionally, we investigated the secretion of Sema4D by various macrophage phenotypes, identifying that M2c macrophages secrete significant levels of Sema4D. This secretion recruited SM22α+SHED as mural cells by inducing endothelial PDGF production on a vasculature-on-a-chip platform, indicating a potential role for macrophages in facilitating vascular stabilization.

Conclusions

Sema4D acts on Plexin-B1, inducing METTL3-mediated PDGF-BB secretion to recruit SHED to stabilize vessels. Macrophages could be a key source of Sema4D for vascular stabilization.

Introduction

A stable and mature vascular network is essential for the survival and function of the engineered constructs, as well as tissue repair in diseases such as ischemic vascular disease and wound healing [1,2,3]. The regeneration of a functional vascular network, which guarantees the supply of O2 and nutrients, remains challenging [1]. Angiogenesis is a complex and sequential process where ECs initiate vascular morphogenesis and then recruit mural cells to stabilize the nascent vessels [4]. The maturation of vessels is a process that transforms an active vascular bed into a quiescent and well-perfused functional network [2, 5]. Without support from mural cells, the nascent vascular tubes are fragile and prone to regress [4]. Therefore, recruiting mural cells to support nascent blood vessels is a critical prerequisite for vessel maturation [6]. The primary mural cells from human tissue are scarcely available, and the variability and limited proliferative capacity of tissue-specific phenotypes would hinder their clinical application [7].

Stem cells from human exfoliated deciduous teeth (SHED) with self-renewal, high proliferation, and multi-potent differentiation capacity are considered promising mesenchymal stem cells for regenerative applications [8]. SHED can be easily harvested from exfoliated deciduous or wisdom teeth without additional injury to donor patients. It is believed that SHED originates from a perivascular microenvironment and shares phenotypic and functional characteristics with mural cells. [9, 10]. Previous studies confirmed that SHED express high levels of mural cell markers, including NG2, PDGFR-β, α-SMA, and SM22α [9, 11]. Xu et al. [12] have successfully induced SHED differentiation into vascular smooth muscle cells (vSMCs). Recently, using a vasculature-on-a-chip device, we demonstrated that SHED could be recruited to enhance vascular stabilization by acting as mural cells [11]. Our results showed that SM22α+SHED lined the abluminal surface of endothelial vessels and were colocalized with the collagen IV positive basement membrane [11], further confirming their mural cell function and, hence, could be a feasible source of mural cells in regenerative applications.

Semaphorin 4D (Sema4D), also known as Cluster of Differentiation 100 (CD100), is a class IV semaphorin family member. Sema4D expression was found in many tissues, including the brain, kidney, heart, bone, and tooth, and has been identified for its roles in immune regulation, axon guidance, tumor progression, angiogenesis, bone remodeling, and tooth development [13,14,15,16]. The angiogenic properties of Sema4D have been reported to be activated through high-affinity receptor Plexin-B1 on endothelial cells [17, 18]. Further studies have shown that tumor-associated macrophages (TAMs)-derived Sema4D contributes to the maturation of tumor vessels by facilitating the recruitment of pericytes through activating Plexin-B1 on endothelial cells [19]. Recently, we [11] demonstrated that Sema4D could act on endothelial Plexin-B1 to enhance angiogenesis by promoting the recruitment of mural cells to stabilize the nascent vessels on an in vitro microfluidic platform. However, in vitro models cannot fully recapitulate the complex interactions of a whole organism, where the physiological functions are achieved in a highly integrated and regulated style [20]. As the blood flow mechanics and perfusion further modulate vascular maturation once the prevascular network is anastomosed with the host blood vessels following transplantation, examining whether Sema4D-mediated mural cell recruitment could give rise to a stable vasculature in vivo is critical. Therefore, in the current study, using the classic in vivo angiogenesis model, Matrigel plug assay, we aimed to study the effects of Sema4D in the recruitment of SHED as mural cells and the formation of mature perfused vessels in vivo [21].

Given the critical role of PDGF-BB in Sema4D and HUVEC-SHED interactions, the mechanism by which Sema4D increases PDGF-BB secretion in HUVECs warrants further investigation [11]. Our previous in vitro work found that Sema4D enhanced the secretion of PDGF-BB in HUVECs. However, Sema4D has no impact on the mRNA expression of PDGF-BB, implying there may be a post-translational regulatory mechanism. Recently, N6-methyladenosine (m6A) has emerged as an important post-translation mechanism that controls multiple cell activities and functions [22]. Methyltransferases, such as METTL3, METTL5, METTL14, and Wilms’ tumor 1-associated protein (WTAP), are involved in m6A methylation. Among them, METTL3 is a key component of the m6A methyltransferase complex that catalyzes the methylation of target transcripts [22, 23]. METTL3-mediated m6A methylation is critical for responding to hypoxic stress and promoting angiogenesis [24,25,26]. Therefore, we also aimed to unravel the downstream signaling mechanism of whether Sema4D enhances PDGF-BB secretion in endothelial cells and vascular stabilization through METTL3-mediated m6A methylation.

Sema4D is expressed by various cells in either membrane-bound or soluble forms, but the paracrine effects of soluble Sema4D are not well understood. Macrophages, which can polarize into M1 or M2 subtypes, are crucial for immune responses and tissue repair during injury and regeneration [27]. M1 macrophages are reported to promote the initial stage of angiogenesis whereas M2 is related to vessel maturation [28, 29]. It has been reported that tumor-associated macrophages (TAMs) promote tumor angiogenesis, vascular maturation, and tumor progression through upregulated expression of Sema4D [19, 30]. M2a and M2c have been reported to induce pericyte recruitment during angiogenesis [28, 29], we asked if macrophages could be a source of Sema4D in guiding vascular stabilization. As only a little is known about Sema4D secretion by macrophages, we also aimed to examine if macrophages could recruit SHED as mural cells via Sema4D-mediated paracrine effects using our previously reported vasculature-on-a-chip model.

Taken together, this study aimed to investigate the effect of Sema4D-Plexin-B1 signaling in mediating endothelial-SHED cross-talk in vascular stabilization in vivo. Additionally, we investigated the downstream mechanism of Sema4D-Plexin-B1 in enhancing endothelial PDGF-BB production and whether M2 macrophages could promote vascular stabilization through Sema4D-mediated paracrine effects.

Methods and materials

Cell culture

SHED were purchased from All Cells (Alameda, CA, USA) and cultured in α-minimum essential medium (α-MEM) supplemented with 10% FBS and 1% penicillin/streptomycin. The mesenchymal origin and multipotent differentiation capacity of the cells were evaluated and published in our previous study [12, 31]. Human umbilical vein endothelial cells (HUVECs) were purchased from ScienCell (Carlsbad, CA, USA) and cultured in endothelial cell medium (ECM, ScienCell) supplemented with 5% (v/v) fetal bovine serum, 1% (v/v) endothelial cell growth supplement, and 1% (v/v) penicillin/streptomycin. HUVECs were cultured on plates coated with 2 μg/cm2 bovine plasma fibronectin (ScienCell). All cell cultures were kept in a 37 °C and 5% CO2 incubator. Passages 4–7 of SHED and passages 3–6 of HUVECs were used in all the downstream experiments.

THP-1 monocytes were a gift from Prof. Lijian Jin and were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. The polarization of THP-1 was done following a published protocol [32]. Briefly, THP-1 cells were differentiated into M0 macrophages with 320 nM PMA treatment for 24 h. M0 macrophages were washed with 1X PBS followed by incubation in media supplemented with IFN-γ (100 ng/mL) and LPS (100 ng/mL) for M1 polarization, IL-4 (40 ng/mL) and IL-13 (20 ng/mL) for M2a polarization and IL-10 (40 ng/mL) for M2c polarization.M0 macrophages and polarized M1, M2a, and M2c macrophages were then cultured in fresh medium for 48 h after which the supernatants were collected, centrifuged to remove any particulates and preserved at − 80 °C for further experiments.

Plexin-B1 knockdown by siRNA

siRNA targeting Plexin-B1 (assay ID: s10702) was purchased from Invitrogen. A siRNA with no target human sequence was used as a negative control. HUVECs were transfected with siRNA (final concentration 50 nM) in Opti‐MEM (Invitrogen) using Lipofectamine™ 3000 reagent (Invitrogen) according to the manufacturer’s instructions. After 24 h, Plexin-B1KD-HUVECs and Negative-HUVECs were used for downstream experiments.

In vivo Matrigel plug angiogenesis assay

The protocol of animal experiments was approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) (Reference No.4969-19). All animal experiments were performed according to the guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health and regulations at the Laboratory Animal Unit of the University of Hong Kong. The work has been reported in line with the ARRIVE guidelines 2.0. Twenty-eight 6–8-week-old male severe combined immunodeficient (SCID/CB17) mice were randomly allocated into seven groups. The allocation of mice is listed in Online Appendix file 1: Table S1. Briefly, HUVECs (or Vector-HUVECs or Plexin-B1KD-HUVECs) and SHED were collected respectively and counted for further use. HUVECs and SHED were mixed at the ratio of 2:1 in the 15 mL tube and centrifuge at 1000 RPM for 5 min. The cells were resuspended with ECM. Then 100 μL cell suspension, containing 2.5 × 106 HUVECs and 1.25 × 106 SHED, was mixed evenly with 400 μL ice-cold growth factor reduced Matrigel (Corning; #356,231). Based on our previous study, Sema4D (1 μg/mL) and PDGFR-β inhibitor (CP-673451, 0.4 μM, MedChemExpress, USA) were premixed with Matrigel for indicated groups [11]. Mice were anesthetized intraperitoneally with xylazine (10 mg/kg) and ketamine (90 mg/kg) (Alfasan, Woerden, The Netherlands). The hair of both lateral hind regions was removed with a shaver. The skin area was wiped with a 70% alcohol pad. Then, 400 μL the cell/Matrigel mixture was injected into the subcutaneous spaces of each side (4 plugs per group). Matrigel plugs were harvested at day 7 or day 14 after euthanization by overdose of Pentobarbital Sodium (Dorminal 20%, Alfasan, Woerden-Holland) via intraperitoneal injection [33]. Blinding was applied when analyzing the results. After harvest, plugs were labeled by numbers and rinsed with PBS immediately and then fixed with 4% paraformaldehyde for 24 h at room temperature. Plugs were oriented before embedding with paraffin to ensure that the skin and muscle layers were visible at the section. Plugs were sectioned vertically at 5 μm thickness. Then, histology and immunofluorescence staining were performed as described below.

Hematoxylin and Eosin (H&E) staining

H&E staining was performed to visualize the blood vessels within the Matrigel plugs. Briefly, the sections were deparaffinized and rehydrated by passing through 100% xylene twice (3 min for each), 100% ethanol twice (1 min for each), 95% ethanol (1 min), 70% ethanol (1 min) and lastly 1 × PBS. The slides were stained with filtered Hematoxylin (Shandon Inc) for 3 min, followed by washing with running tap water for 3 min. Subsequently, the slides were differentiated in 0.1% HCl (in 70% ethanol) for 3 s and then rinsed with running tap water. Then, the sections were placed in a bluing reagent (1% ammonia water) for 1 min and the washing step was repeated. After that, the slides were immersed in filtered Eosin for 50 s and washed for 30 s. After mounting, sections were imaged under an inverted microscope (Nikon Eclipse LV100N POL, Tokyo, Japan) at 20 × and 50 × magnifications.

Immunofluorescence staining for Matrigel section

Immunofluorescence staining was conducted to examine the interaction between HUVECs and SHED on the blood vessels formed within Matrigel plugs. Briefly, after the deparaffinization and rehydration process, the slides were immersed in 1 × antigen retrieval solution (Dako, Copenhagen, Denmark) at 95 °C for 20 min. After cooling down, the slides were washed with 1 × PBS. Then, the sections were blocked in a blocking buffer (1 × PBS, 5% BSA, 0.3% Triton X-100) for 1 h at room temperature. The sections were incubated with primary antibody (mouse anti-human CD31, Cell signaling, 1:300; rabbit anti-SM22α, Abcam, 1:300; rabbit anti-human NG2, Cell signaling, 1:200; rabbit anti-PDGFR-β, Abcam, 1:100; mouse monoclonal anti-Collagen IV-Alexa Fluor™ 647 (eBioscience™, 10 μg/mL); rabbit IgG isotype control, Invitrogen, 1:2000; mouse IgG isotype control, Abcam,1:1000) diluted in blocking buffer at 4 °C overnight in a humidity tray containing distilled water. After washing with 1 × PBST (1 × PBS + 0.1% Tween20) three times, the sections were incubated with secondary antibody (Alexa Fluor® 488 Conjugated goat anti-mouse IgG, Cell Signaling, 1:500; Alexa Fluor® 555 Conjugated goat anti-rabbit IgG, Invitrogen, 1:500) for 1 h at room temperature. Cell nuclei were stained with DAPI (Sigma-Aldrich) or DRAQ5 (Thermo Scientific). Images were captured under a Zeiss LSM900 confocal microscope with Airyscan laser (Carl Zeiss, Germany) at 20 × , 40 × and 63 × magnification.

Quantification of blood vessels

Blinding was performed to group allocation during data collection and analysis. Five random areas per sample were captured at 20× (an area of 0.295 mm2) to quantify the newly formed functional blood vessels. Perfused vessels were identified as lumens filled with red blood cells in H&E-stained sections. The total number of perfused vessels was counted in the five randomly selected areas, and the mean number per sample was calculated. Accordingly, sections from four different samples were examined to obtain the mean for each group based on these four samples. To examine the recruitment of SHED as mural cells on HUVEC-formed vasculature, the number of SM22α+SHED covered vessels (CD31 +) was quantified in immunofluorescence-stained sections using Image J software (National Institutes of Health, Bethesda, MD).

RNA m6A quantification

Total RNA was extracted from HUVECs via Yishen Quick RNA Extraction Kit (RightGene) and quantified using a Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA). The m6A modification level of total RNA was examined by EpiQuik m6A RNA Methylation Quantification Kit (Epigentek Group Inc., Farmingdale, NY, USA) according to the manufacturer’s instructions. Briefly, 200 ng RNA were coated on assay wells, followed by capture antibody solution and detection antibody solution. The m6A levels were quantified colorimetrically by microplate reader.

Quantitative real-time PCR

Total RNA was isolated from HUVECs with Yishen Quick RNA Extraction Kit (RightGene) and then reverse‐transcribed into cDNA with PrimeScript® RT reagent kit (Takara). Afterward, qRT-PCR was performed with TBGREEN Premix Ex TaqTM (Tsingke). Analysis was performed on a qRT-PCR System (Bio‐Rad). The housekeeping gene β-Actin was used for normalization. The primers used in this study are shown in Online Appendix file 1: Table S2.

METTL3 Knockdown by siRNA

siRNA targeting METTL3 (assay ID: 132,906) was purchased from Invitrogen. A siRNA with no target human sequence was used as a negative control. HUVECs were transfected with siRNA (final concentration 50 nM) in Opti‐MEM (Invitrogen) using Lipofectamine™ 3000 reagent (Invitrogen) according to the manufacturer’s instructions. After 24 h, METTL3 knockdown HUVECs (METTL3KD-HUVECs) and Negative-HUVECs (HUVECs) were used for downstream experiments. Briefly, HUVECs were seeded on 6 well plates at a density of 3 × 105 cells per well until they reached 80% confluence. Then HUVECs were treated with si-negtive control (si-NC) or si-METTL3 for 24 h, followed by treated with Sema4D (1 μg/mL) for further 24 h.

Western blotting

Total protein was extracted from HUVECs using a radio-immunoprecipitation assay lysis buffer (RIPA) with protease and phosphatase inhibitors (Cwbio, Beijing, China). 30μL protein lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel (Vazyme) electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore). Then, the membranes were blocked with 5% non-fat milk for 1 h at room temperature. Subsequently, they were incubated overnight at 4 °C with the following primary antibodies: monoclonal Mouse anti-METTL3 (1:1000, Santa Cruze), polyclonal rabbit anti-PGDF-BB (1:1000, Abcam), polyclonal rabbit anti-GAPDH or polyclonal rabbit anti-β-ACTIN (1:1000, Cell Signal Technology). After washing with tris-buffered saline and tween 20 (TBST), the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) for 1 h at room temperature. After repeating the washing step, the target protein signal was measured by enhanced chemiluminescence (Thermo Scientific). Then, the membranes were visualized using a gel imaging system (Bio-Rad, Hercules, CA). The expression of protein was quantified by ImageJ software.

ELISA

The secretory protein levels of PDGF-BB and Sema4D were examined using respective ELISA kits (RayBiotech, Inc., Norcross, GA, U.S.A.; R&D Systems, Minnesota, US). The preparation of METTL3KD-HUVECs were described above. Supernatants were collected from negative HUVECs and METTL3KD-HUVECs after treated with Sema4D for 24 h. Besides, PDGF-BB ELISA were also performed to detect the effect of macrophage conditioned medium on HUVECs. Briefly, HUVECs were seeded on 6 well plates at a density of 3 × 105 cells per well until they reached 80% confluence and treated with M0-CM, M2c-CM, Sema4D (1 μg/mL) and M2c-CM supplemented with Pepinimab (20 μg/mL, MedchemExpress, USA) which is a Sema4D inhibitor. Supernatants were collected after culturing for 24 h and centrifuged to remove any particulates, following which ELISA was performed according to the manufacturer’s instructions. For Sema4D ELISA, THP-1cells were seeded on a 6 cm dish at a density of 4 × 106 cells and polarized into different subtypes of macrophages as described above. After polarization, a fresh medium was added and cultured for 48 h. Then, the supernatant was collected and centrifuged to remove any particulates. The conditioned medium (M0-CM, M1-CM, M2a-CM, M2c-CM) was stored at -80 refrigerator immediately for further use.

Angiogenesis assay using 3D vasculature-on-a-chip microfluidic device

AIM Biotech 3D Cell Culture Chips (AIM Biotech, Ayer Rajah Crescent, Singapore) were used to investigate the interaction between HUVECs and SHED under different treatments. Briefly, HUVECs or METTL3KD-HUVECs were encapsulated within fibrinogen solution at the final concentration of 4 × 106 cells/mL and 2.5 mg/mL fibrinogen with 0.15 U/mL aprotinin (Sigma-Aldrich). The fibrin/cell suspension was then mixed with thrombin (Sigma-Aldrich) and injected into the central channel immediately. The upper channel was coated with bovine plasma fibronectin (30 μg/mL, ScienCell) for 1 h and seeded with HUVECs or METTL3KD-HUVECs at a concentration of 2 × 106 cells/mL. After HUVECs formed a vascular network within the gel, the SHED was seeded in the lower channel at 2 × 106 cells/mL concentration. At the same time, Sema4D (1 μg/mL), M0-CM and M2c-CM with or without PDGFR-β inhibitor, Sema4D inhibitor were added into the channels. The culture medium of each channel was refreshed daily. The samples were fixed and stained on day 7.

Immunofluorescence staining on microfluidic model

Samples were washed with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min. Then, samples were blocked with 5% bovine serum albumin (BSA, Beyotime Institute of Biotechnology, China) containing 0.2% (v/v) Triton-X100 for 1 h at room temperature. Mouse monoclonal anti-CD31 antibody (Cell Signaling Technology, Danvers, MA, USA,1:300) and rabbit polyclonal anti-SM22α antibody (Abcam, Cambridge, MA, USA,1:300) were used in immunofluorescence staining. Alexa Fluor® 488 Conjugated goat anti-mouse IgG (Cell Signaling Technology) and Alexa Fluor® 555 Conjugated goat anti-rabbit IgG (Invitrogen) were used as secondary antibodies. Cell nuclei were stained with hoechst (Invitrogen, 2 μg/mL). The images were captured under a Zeiss LSM 900 confocal microscope with an Airyscan laser (Carl Zeiss, Germany). Z-stack images were used for statistic. The SM22α+SHED coverage was defined as the ratio of SM22α+SHED covered vessel length to total vessel length. The Image J software were used to quantified the length (National Institutes of Health, Bethesda, MD).

Statistical analysis

The experiments were performed in triplicate with three independent experiments. All data are shown as mean ± standard deviation (SD). Statistical analyses were carried out using Student’s t-test or one-way analysis of variance with Tukey’s post hoc test to determine significant differences between groups. All analyses were performed using the GraphPad Prism 8 software (GraphPad Software, Inc., San Diego, CA). P < 0.05 was considered statistically significant.

Results

Sema4D enhances vessel formation and the percentage of SM22α+SHED covered vessels by acting on Plexin-B1 on HUVECs in vivo

To investigate the effect of Sema4D on SHED-supported vessel formation and stabilization, the in vivo Matrigel plug angiogenesis assay was performed. After 7 and 14 days of implantation, macroscopic and H&E staining analyses showed that Sema4D significantly increased vascularization in HUVECs + SHED Matrigel plugs, as evidenced by the highest number of perfused blood vessels (Fig. 1A, B, E, G). When Plexin-B1 was knocked down on HUVECs, Sema4D failed to show any significant enhancement in the number of perfused vessels. Sema4D treated HUVECs + SHED group showed a significantly higher number of SM22α+SHED covered vessels compared to the control group at both 7 (Fig. 1C, F) and 14 days (Fig. 1D, H) of implantation (Online Appendix file 1: Figure S1) as shown by immunofluorescent staining for CD31 and SM22α. When Plexin-B1 was knocked down on HUVECs, the effect of Sema4D on the number of SM22α+SHED covered vessels significantly decreased at both 7 and 14 days of implantation. Nonimmune immunoglobulins of the same isotype were used as negative controls (Online Appendix file 1: Figure S2).

Fig. 1
figure 1

Sema4D enhances vessel formation and stabilization by acting on the Plexin-B1 receptor on HUVECs in vivo. A, B Representative images and H&E-stained sections of Matrigel plugs retrieved from SCID mice at day 7 and day 14, scale bar = 100 μm. C, D Representative images of immunofluorescence-stained sections for CD31 and SM22α at day 7 and day 14. The arrows show that SM22α+SHED lining the abluminal surface of endothelial vessels formed by CD31+HUVECs, scale bar = 50 μm. E, G Quantification of the number of perfused blood vessels from H&E-staining at day 7 and day 14. F, H Quantification of the percentage of SM22α.+SHED covered vessels from immunofluorescence staining at day 7 and day 14. * P < 0.05, ** P < 0.01

Full size image

In addition to SM22α, immunofluorescence for NG2 and PDGFR-β also confirmed the mural cell-like phenotype of SHED (Fig. 2A, B). The triple immunofluorescent staining for CD31, SM22α, and Collagen IV allowed for better visualization of the structural organization of the newly formed blood vessels. SM22α+SHED were localized on the abluminal surface of CD31+ endothelial lined vessel lumens. Both SM22α+SHED and CD31+HUVECs were co-localized with the collagen IV-positive basement membrane (Fig. 2C). This arrangement indicates that SM22α+SHED were effectively integrated into the vessel wall and were functioning as mural cells, supporting the structure of the blood vessels in vivo.

Fig. 2
figure 2

Representative images of immunofluorescent staining to demonstrate the mural cell-like role of SHED. A, B Representative images of immunofluorescent stained sections for NG2 or PDGFR-β and CD31 at day 7. The arrows indicate that the vessels formed by CD31 + HUVECs were supported by SM22α + SHED. The white triangles indicate the vessels without SM22α+SHED support. * marks the vessel lumens. Scale bar = 50 μm. C Triple immunofluorescent staining for CD31, SM22α, and Collagen IV of Matrigel plug sections at day 7. The arrows indicate that vascular lumens lined by CD31+HUVECs were supported by SM22α+SHED on the abluminal surface and embedded within the collagen IV + basement membrane (pink). The white triangles indicate the vessels without SM22α+SHED support. * marks the vessel lumens, scale bar = 20 μm

Full size image

Sema4D enhances vessel formation, and the percentage of SM22α+SHED covered vessels via PDGF-BB/ PDGFR-β axis in vivo

Our previous in vitro work demonstrated that Sema4D could enhance vessel formation and stabilization through endothelial-derived PDGF-BB by recruiting SHED as mural cells [11]. Consistently, the in vivo Matrigel plug assay showed that Sema4D significantly increased the number of perfused vessels. However, when PDGFR-β inhibitor was added in the Sema4D-treated HUVECs + SHED group, the number of perfused vessels significantly dropped at both 7 and 14 days of implantation (Fig. 3A, B, E, G). Accordingly, the addition of PDGFR-β inhibitor significantly reduced the percentage of SM22α+SHED covered vessels at both day 7 and 14, indicating an indispensable role of PDGF-BB in mediating SHED-supported vascular stabilization (Fig. 3C, D, F, H, Online Appendix file 1: Figure S3).

Fig. 3
figure 3

PDGFR-β inhibitor suppressed Sema4D-induced vessel formation and stabilization in vivo. A, B Representative images of Matrigel plugs and H&E-stained sections of Matrigel plugs retrieved from SCID mice at day 7 and day 14, scale bar = 100 μm. C, D Representative images of immunofluorescence-stained sections for CD31 and SM22α at day 7 and day 14. The arrows indicate that SM22α+SHED lining the abluminal surface of endothelial vessels formed by CD31+HUVECs, scale bar = 50 μm. E, G Quantification of the number of perfused blood vessels from H&E-staining on days 7 and 14. F, H Quantification of the number of SM22α.+SHED covered vessels from immunofluorescence staining at day 7 and day 14. * P < 0.05, ** P < 0.01

Full size image

Sema4D induces METTL3-mediated N6-methyladenosine methylation in HUVECs to regulate PDGF-BB secretion and facilitate SHED-supported vascular stabilization

To investigate the downstream mechanism of Sema4D-induced endothelial-derived PDGF-BB, we detected the m6A modification level of total RNA and the expression of relative methylation genes in Sema4D-treated HUVECs. The results showed that Sema4D increased m6A methylation levels in HUVECs at 12 and 24 h (Fig. 4A). The qRT-PCR results showed a significant increase in METTL3, METTL14, and METTL5 expression after being treated with Sema4D for 24 h (Fig. 4B, Online Appendix file 1: Figure S4A). Additionally, western blot analysis revealed Sema4D significantly increased in the expression of METTL3, a key component of the m6A methyltransferase complex, after cultivating for 24 h (Fig. 4C). METTL3 was knocked down in HUVECs via siRNA, and the effect was confirmed by qRT-PCR and western blotting (Online Appendix file 1: Figure S4B). Western blotting results showed that the expression of PDGF-BB in Sema4D-treated METTL3KD-HUVECs was significantly decreased compared with that in Sema4D-treated HUVECs (Fig. 4D). ELISA results revealed significantly lower levels of PDGF-BB in METTL3KD-HUVECs than HUVECs when treated with Sema4D (Fig. 4E). To verify the function of METTL3 in Sema4D-induced SHED-supported vessel formation and stabilization, a vasculature-on-a-chip model was used (Online Appendix file 1: Figure S5A&B). The knockdown of METTL3 in HUVECs resulted in significantly reduced SM22α+SHED covered vessels under Sema4D treatment, indicating that Sema4D recruits SHED as mural cells in vascular stabilization via METTL3-induced m6A modification (Fig. 4F). Taken together, METTL3-mediated N6-methyladenosine methylation could be the downstream mechanism of Sema4D in upregulating HUVECs-derived PDGF-BB and facilitating SHED-supported vascular stabilization.

Fig. 4
figure 4

METTL3 knockdown in HUVECs impaired Sema4D-induced vessel stabilization. A Sema4D increased the m6A modification level in HUVECs at 12 h and 24 h. B The expression of METTL3, METTL14 and METTL5 in HUVECs with Sema4D treatment for 24 h. C Western blotting results of METTL3 in HUVECs after cultivating with Sema4D for different durations. Full-length blots are presented in Appendix file 2: Fig. 4C. D METTL3 knockdown in HUVECs decreases the expression of PDGF-BB in the presence of Sema4D. Full-length blots are presented in appendix file 2: Fig. 4D. E METTL3 knockdown in HUVECs downregulates the secretion of PDGF-BB in the presence of Sema4D for 24 h. F Representative Z-stack images and quantified SM22α+SHED coverage of the microfluidic assay of HUVECs or METTL3KD-HUVECs and SHED in the presence of Sema4D. Scale bar = 100 μm. * P < 0.05, ** P < 0.01

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Macrophages are an important source of Sema4D to trigger PDGF-BB secretion in HUVECs and enhance vascular stabilization

To investigate whether macrophages could be an endogenous source of Sema4D, ELISA was performed to detect the Sema4D levels in different macrophage phenotypes. The results showed that M0 and M2c phenotypes have higher expression of Sema4D (Fig. 5A). In addition, significantly high PDGF-BB levels were detected in HUVECs treated with M0 and M2c conditioned medium (CM) for 24 h (Fig. 5B). Further, the addition of Sema4D inhibitor (Pepinimab) significantly reduced the effect of M2c-CM-secreted PDGF-BB levels in HUVECs (Fig. 5C). This confirmed that Sema4D in M2c-CM is responsible for the significantly increased PDGF-BB levels in Macrophage CM-treated HUVECs. The results of the vasculature-on-a-chip model (Online Appendix file 1: Figure S5C) showed that M2c-CM significantly upregulated the percentage of SM22α+SHED covered vascular networks (Fig. 5D), which is similar to Sema4D treated groups. The addition of Sema4D inhibitor and PDGFR-β inhibitor (CP-673451) (Online Appendix file 1: Figure S5D) resulted in significantly reduced SM22α+SHED covered vessels in M2c-CM added groups (Fig. 5E). This confirmed the effect of M2c-CM on vascular stabilization, which is mediated through Sema4D/PDGF-BB/PDGFR signaling.

Fig. 5
figure 5

M2c enhances vascular stabilization via paracrine effects. A The secretion of Sema4D in different phenotypes of macrophages for 48 h. B PDGF-BB expression in macrophage conditioned medium treated HUVECs for 24 h. Sema4D was used as a positive control. C The expression of PDGF-BB in M2c macrophage CM-treated HUVECs with and without Sema4D inhibitor. D Representative orthogonal projection images and quantified SM22α+SHED coverage of the microfluidic assay treated with M0-CM or M2c-CM or Sema4D. E Representative orthogonal projection images and quantified SM22α.+SHED coverage of the microfluidic assay treated with M2c-CM with or without Sema4D inhibitor or PDGFR-β inhibitor. Scale bar = 100 μm. * P < 0.05, ** P < 0.01

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Discussion

Semaphorins are membrane-bound and soluble proteins essential for regulating cellular functions and interactions. The family has eight classes, with classes 3–7 found in vertebrates [34]. Sema4D, Sema6A, Sema6D, and Sema7A have been identified as pro-angiogenic semaphorins, while Sema3A, Sema3B, Sema3D, Sema3E, and Sema3F are recognized as anti-angiogenic [34, 35]. Sema4D has been shown to enhance the maturation of tumor vessels through recruiting pericytes [19]. Additionally, Sema4D has synergistic effects with VEGF in promoting angiogenesis [18]; and induces endothelial cell chemotaxis and tube formation through the highest-affinity receptor Plexin-B1 [17, 36]. Recently, Sema4D has also been reported to promote the polarization of macrophages to M2 by reducing the expression of IL-1β, IL-6, and TNF-α [37]. We recently showed that Sema4D promotes vascular stabilization by recruiting dental stem cells as mural cells through endothelial-derived PDGF-BB on a vasculature-on-a-chip model in vitro [11]. Qi et al. reported that PDGF-BB exerts paracrine effects on the migration and proliferation of vSMCs by ECs in the coculture flow chamber system [38]. In this study, our in vivo results showed that Sema4D-Plexin-B1 signaling enhances vascular stabilization through endothelial-derived PDGF-BB. Inhibition of Sema4D effects by knocking down Plexin-B1 on HUVECs and blocking PDGFR-β on SEHD significantly reduced the vascular formation and stabilization. This, in turn, proves that the in vitro vasculature-on-a-chip model successfully assesses vascular stabilization.

Methylation of m6A is the most prevalent internal modification of mammalian RNAs, which regulates RNA stability or translation [22]. METTL3 is a key component of the m6A methyltransferase complex that catalyzes the methylation of target transcripts and has been implicated in various pathological and physiological processes [22, 23]. In physiological processes, METTL3-mediated m6A methylation is critical for responding to hypoxic stress and promoting angiogenesis by different signaling, including Wnt and PI3K/AKT signaling pathways [24,25,26]. METTL3 has also been implicated in tumor angiogenesis and metastasis by enhancing cancer cell glycolysis and proliferation [23]. METTL3 promotes pathological angiogenesis by targeting EphA2, VEGFA, and HDGF through different mechanisms involving PI3K/AKT and ERK1/2 signaling pathways and JAK2/STAT3 signaling pathways [39,40,41]. A previous study showed that METTL3 is essential for EC function and angiogenesis by regulating m6A RNA methylation in ECs, potentially through influencing let-7e and miR-17-92 cluster processing [42]. We observed that Sema4D promoted the expression of METTL3, METTL5, and METTL14 in HUVECs, and silencing METTL3 inhibited the upregulation of PDGF-BB by Sema4D, suggesting that METTL3 may mediate the Sema4D-induced promotion of PDGF-BB secretion. Furthermore, the results of the vasculature-on-a-chip model showed that silencing METTL3 significantly blocked the effect of Sema4D on vascular stabilization. Therefore, METTL3-mediated m6A methylation could be the downstream signaling mechanism of Sema4D on the upregulation of PDGF-BB secretion and vascular stabilization.

The interaction between macrophages and endothelial cells plays essential roles in physiological and pathological conditions by regulating inflammation, vascularization, and tissue remodeling [43]. Macrophages have been reported to support endothelium integrity by regulating the metabolism and function of endothelial cells [27]. The main mechanism for the pro-angiogenic effects of macrophages is thought to be executed via secreting growth factors. Besides, other mechanisms, such as function as endothelial cell chaperones and transdifferentiation into support cells, were also reported [44]. M1 macrophages are reported to promote the initial stage of angiogenesis via the upregulated secretion of VEGF, whereas M2 macrophages play a critical role during vessel maturation by expression of PDGF-BB, TGF-β1, and MMP9 [28, 29]. It has been reported that PDGF-BB, not TGF-β1, is essential for vSMC recruitment and proliferation in the Coculture Flow Chamber System [38]. For the first time, our study demonstrated that M2c could be an endogenous source of Sema4D that contributes to vascular maturation by inducing endothelial PDGF-BB production on a vasculature-on-a-chip platform. This indicates a potential role for macrophages in facilitating vascular stabilization through Sema4D-mediated paracrine signaling. Figure S6 (Online Appendix file 1) shows the proposed role of the M2c macrophage-Sema4D-Plexin-B1-METTL3-PDGF-BB signaling axis in vascular stabilization.

However, further studies are needed to confirm the methylation-related effects of Sema4D-Plexin-B1-METTL3 signaling to investigate and confirm whether METTL3 directly binds to the promoter region of the target genes and regulates their transcription levels in the context of Sema4D stimulation. In addition, further studies are needed to demonstrate how host macrophage polarization can be induced to achieve vascular stabilization in engineered tissue constructs.

Conclusion

This study showed that Sema4D-Plexin-B1 signaling enhances the formation of a perfused and mural cell-supported vasculature through endothelial-derived PDGF-BB in vivo. Additionally, we discovered that Sema4D-PlexinB1 signaling could activate downstream methylation signaling through METTL3 to enhance endothelial PDGF production. Furthermore, we demonstrated that M2c macrophages promote vascular stabilization through Sema4D by endothelial-derived PDGF-BB in a 3D microfluidic model. Therefore, macrophages could act as an endogenous source of Sema4D that could be modulated in vascular stabilization in engineered tissue constructs. This could be a new therapeutic target for tissue repair and regeneration.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.

Abbreviations

α-MEM:

α-Minimum essential medium

CD100:

Cluster of differentiation 100

CM:

Conditioned medium

ECM:

Endothelial cell medium

H&E:

Hematoxylin and Eosin

HUVECs:

Human umbilical vein endothelial cells

m6A:

N6-methyladenosine

METTL3:

Methyltransferase-like 3

PDGF-BB:

Platelet-derived growth factor-BB

Sema4D:

Semaphorin 4D

SHED:

Stem cells from human exfoliated deciduous teeth

SM22α+SHED:

SM22α-positive SHED

TAMs:

Tumor-associated macrophages

vSMCs:

Vascular smooth muscle cells

WTAP:

Wilms’ tumor 1-associated protein

References

  1. Chen X, Yu W, Zhang J, Fan X, Liu X, Liu Q, Pan S, Dixon RAF, Li P, Yu P, et al. Therapeutic angiogenesis and tissue revascularization in ischemic vascular disease. J Biol Eng. 2023;17(1):13.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Rouwkema J, Khademhosseini A. Vascularization and angiogenesis in tissue engineering: beyond creating static networks. Trends Biotechnol. 2016;34(9):733–45.

    Article  CAS  PubMed  Google Scholar 

  3. Ben-Shaul S, Landau S, Merdler U, Levenberg S. Mature vessel networks in engineered tissue promote graft-host anastomosis and prevent graft thrombosis. Proc Natl Acad Sci USA. 2019;116(8):2955–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Gerhardt H, Betsholtz C. Endothelial-pericyte interactions in angiogenesis. Cell Tissue Res. 2003;314(1):15–23.

    Article  PubMed  Google Scholar 

  5. Zanotelli MR, Ardalani H, Zhang J, Hou Z, Nguyen EH, Swanson S, Nguyen BK, Bolin J, Elwell A, Bischel LL, et al. Stable engineered vascular networks from human induced pluripotent stem cell-derived endothelial cells cultured in synthetic hydrogels. Acta Biomater. 2016;35:32–41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Traktuev DO, Merfeld-Clauss S, Li J, Kolonin M, Arap W, Pasqualini R, Johnstone BH, March KL. A population of multipotent CD34-positive adipose stromal cells share pericyte and mesenchymal surface markers, reside in a periendothelial location, and stabilize endothelial networks. Circ Res. 2008;102(1):77–85.

    Article  CAS  PubMed  Google Scholar 

  7. Kim S, Von Recum H. Endothelial stem cells and precursors for tissue engineering: cell source, differentiation, selection, and application. Tissue Eng Part B Rev. 2008;14(1):133–47.

    Article  CAS  PubMed  Google Scholar 

  8. Han Y, Zhang L, Zhang C, Dissanayaka WL. Guiding lineage specific differentiation of SHED for target tissue/organ regeneration. Curr Stem Cell Res Ther. 2021;16(5):518–34.

    Article  CAS  PubMed  Google Scholar 

  9. Zhu SY, Yuan CY, Lin YF, Liu H, Yang YQ, Wong HM, Zhang CF, Wang PL, Gu M. Stem Cells from Human Exfoliated Deciduous Teeth (SHEDs) and Dental Pulp Stem Cells (DPSCs) Display a Similar Profile with Pericytes. Stem Cells Int. 2021;2021:8859902.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, Shi S. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci USA. 2003;100(10):5807–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zhang L, Han Y, Chen Q, Dissanayaka WL. Sema4D-plexin-B1 signaling in recruiting dental stem cells for vascular stabilization on a microfluidic platform. Lab Chip. 2022;22(23):4632–44.

    Article  CAS  PubMed  Google Scholar 

  12. Xu J, Zhu S, Heng B, Dissanayaka WL, Zhang C. TGF-β1-induced differentiation of SHED into functional smooth muscle cells. Stem Cell Res Ther. 2017;8(1):10.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Zeng Y, Wang L, Liu L, Wang M, Yan L, Ye L, Song D, Huang D. The potential immunomodulatory roles of semaphorin 4D in human periapical lesions. J Endod. 2023;49(1):62–8.

    Article  PubMed  Google Scholar 

  14. Otsu K, Ida-Yonemochi H, Fujiwara N, Harada H. The semaphorin 4D-RhoA-Akt signal cascade regulates enamel matrix secretion in coordination with cell polarization during ameloblast differentiation. J Bone Miner Res. 2016;31(11):1943–54.

    Article  CAS  PubMed  Google Scholar 

  15. Shrestha A. Semaphorin 3A and class 4 semaphorins in tooth innervation and development. 2014.

  16. Maleki KT, Cornillet M, Björkström NK. Soluble SEMA4D/CD100: a novel immunoregulator in infectious and inflammatory diseases. Clin Immunol. 2016;163:52–9.

    Article  CAS  PubMed  Google Scholar 

  17. Conrotto P, Valdembri D, Corso S, Serini G, Tamagnone L, Comoglio PM, Bussolino F, Giordano S. Sema4D induces angiogenesis through Met recruitment by Plexin B1. Blood. 2005;105(11):4321–9.

    Article  CAS  PubMed  Google Scholar 

  18. Chen Y, Zhang L. Liu W-x, Wang K: VEGF and SEMA4D have synergistic effects on the promotion of angiogenesis in epithelial ovarian cancer. Cell Mol Biol Lett. 2018;23(1):2.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Sierra JR, Corso S, Caione L, Cepero V, Conrotto P, Cignetti A, Piacibello W, Kumanogoh A, Kikutani H, Comoglio PM. Tumor angiogenesis and progression are enhanced by Sema4D produced by tumor-associated macrophages. J Exp Med. 2008;205(7):1673–85.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Barré-Sinoussi F, Montagutelli X. Animal models are essential to biological research: issues and perspectives. Future Sci OA. 2015;1(4):Fso63.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Kastana P, Zahra FT, Ntenekou D, Katraki-Pavlou S, Beis D, Lionakis MS, Mikelis CM, Papadimitriou E. Matrigel plug assay for in vivo evaluation of angiogenesis. Methods Mol Biol. 2019;1952:219–32.

    Article  CAS  PubMed  Google Scholar 

  22. Liu S, Zhuo L, Wang J, Zhang Q, Li Q, Li G, Yan L, Jin T, Pan T, Sui X, et al. METTL3 plays multiple functions in biological processes. Am J Cancer Res. 2020;10(6):1631–46.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Yue S-W, Liu H-L, Su H-F, Luo C, Liang H-F, Zhang B-X, Zhang W. m6A-regulated tumor glycolysis: new advances in epigenetics and metabolism. Mol Cancer. 2023;22(1):137.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Yao M-D, Jiang Q, Ma Y, Liu C, Zhu C-Y, Sun Y-N, Shan K, Ge H-M, Zhang Q-Y, Zhang H-Y, et al. Role of METTL3-dependent N6-methyladenosine mRNA modification in the promotion of angiogenesis. Mol Ther. 2020;28(10):2191–202.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Jiang W, Zhu P, Huang F, Zhao Z, Zhang T, An X, Liao F, Guo L, Liu Y, Zhou N, et al. The RNA methyltransferase METTL3 promotes endothelial progenitor cell angiogenesis in mandibular distraction osteogenesis via the PI3K/AKT pathway. Front Cell Dev Biol. 2021;9:720925.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Tian C, Huang Y, Li Q, Feng Z, Qiong X. Mettl3 regulates osteogenic differentiation and alternative splicing of vegfa in bone marrow mesenchymal stem cells. Int J Mol Sci. 2019;20(3):551. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ijms20030551.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hong H, Tian XY. The role of macrophages in vascular repair and regeneration after ischemic injury. Int J Mol Sci. 2020;21(17):6328. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ijms21176328.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Graney PL, Ben-Shaul S, Landau S, Bajpai A, Singh B, Eager J, Cohen A, Levenberg S, Spiller KL. Macrophages of diverse phenotypes drive vascularization of engineered tissues. Sci Adv. 2020;6(18):eaay6391.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Spiller KL, Anfang RR, Spiller KJ, Ng J, Nakazawa KR, Daulton JW, Vunjak-Novakovic G. The role of macrophage phenotype in vascularization of tissue engineering scaffolds. Biomaterials. 2014;35(15):4477–88.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Li H, Wang JS, Mu LJ, Shan KS, Li LP, Zhou YB. Promotion of Sema4D expression by tumor-associated macrophages: Significance in gastric carcinoma. World J Gastroenterol. 2018;24(5):593–601.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Han Y, Koohi-Moghadam M, Chen Q, Zhang L, Chopra H, Zhang J, Dissanayaka WL. HIF-1α stabilization boosts pulp regeneration by modulating cell metabolism. J Dent Res. 2022;101(10):1214–26.

    Article  CAS  PubMed  Google Scholar 

  32. Baxter EW, Graham A, Re NA, Carr IM, Robinson JI, Mackie SL, Morgan AW. Standardized protocols for differentiation of THP-1 cells to macrophages with distinct M(IFNγ+LPS), M(IL-4) and M(IL-10) phenotypes. J Immunol Methods. 2020;478:112721.

    Article  CAS  PubMed  Google Scholar 

  33. Yuan K, Orcholski ME, Huang NF, de Jesus Perez VA. In vivo study of human endothelial-pericyte interaction using the matrix gel plug assay in mouse. J Visua Exp. 2016. https://doiorg.publicaciones.saludcastillayleon.es/10.3791/54617.

    Article  Google Scholar 

  34. Iragavarapu-Charyulu V, Wojcikiewicz E, Urdaneta A. Semaphorins in angiogenesis and autoimmune diseases: Therapeutic targets? Front Immunol. 2020;11:346.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Bica C, Tirpe A, Nutu A, Ciocan C, Chira S, Gurzau ES, Braicu C, Berindan-Neagoe I. Emerging roles and mechanisms of semaphorins activity in cancer. Life Sci. 2023;318:121499.

    Article  CAS  PubMed  Google Scholar 

  36. Oinuma I, Ito Y, Katoh H, Negishi M. Semaphorin 4D/Plexin-B1 stimulates PTEN activity through R-Ras GTPase-activating protein activity, inducing growth cone collapse in hippocampal neurons. J Biol Chem. 2010;285(36):28200–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Zhou JY, Mei YK, Qian XN, Yao ZH, Zhu YW, Wei YW, Qiu J. Modulation of SEMA4D-modified titanium surface on M2 macrophage polarization via activation of Rho/ROCK-mediated lactate release of endothelial cells: in vitro and in vivo. Colloids Surf B Biointerfaces. 2024;234:113691.

    Article  CAS  PubMed  Google Scholar 

  38. Qi YX, Jiang J, Jiang XH, Wang XD, Ji SY, Han Y, Long DK, Shen BR, Yan ZQ, Chien S, et al. PDGF-BB and TGF-{beta}1 on cross-talk between endothelial and smooth muscle cells in vascular remodeling induced by low shear stress. Proc Natl Acad Sci USA. 2011;108(5):1908–13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Liu X, He H, Zhang F, Hu X, Bi F, Li K, Yu H, Zhao Y, Teng X, Li J, et al. m6A methylated EphA2 and VEGFA through IGF2BP2/3 regulation promotes vasculogenic mimicry in colorectal cancer via PI3K/AKT and ERK1/2 signaling. Cell Death Dis. 2022;13(5):483.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Dong G, Yu J, Shan G, Su L, Yu N, Yang S. N6-methyladenosine methyltransferase METTL3 promotes angiogenesis and atherosclerosis by upregulating the JAK2/STAT3 pathway via m6A reader IGF2BP1. Front Cell Dev Biol. 2021;9:731810.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Wang Q, Chen C, Ding Q, Zhao Y, Wang Z, Chen J, Jiang Z, Zhang Y, Xu G, Zhang J, et al. METTL3-mediated m6A modification of HDGF mRNA promotes gastric cancer progression and has prognostic significance. Gut. 2020;69(7):1193–205.

    Article  CAS  PubMed  Google Scholar 

  42. Chamorro-Jorganes A, Sweaad WK, Katare R, Besnier M, Anwar M, Beazley-Long N, Sala-Newby G, Ruiz-Polo I, Chandrasekera D, Ritchie AA, et al. METTL3 regulates angiogenesis by modulating let-7e-5p and miRNA-18a-5p expression in endothelial cells. Arterioscler Thromb Vasc Biol. 2021;41(6):e325–37.

    Article  CAS  PubMed  Google Scholar 

  43. Guan Y, Racioppi L, Gerecht S. Engineering biomaterials to tailor the microenvironment for macrophage–endothelium interactions. Nat Rev Mater. 2023;8(10):688–99.

    Article  CAS  Google Scholar 

  44. Nolfi AL, Behun MN, Yates CC, Brown BN, Kulkarni M. Beyond growth factors: macrophage-centric strategies for angiogenesis. Curr Pathobiol Rep. 2020;8:111–20.

    Article  Google Scholar 

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Acknowledgements

The authors declare that they have not used AI-generated work in this manuscript.

Funding

This research was supported by the Research Grants Council, Hong Kong (General Research Fund; 17117619).

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Authors and Affiliations

  1. Hospital of Stomatology, Guanghua School of Stomatology, Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, 510055, Guangdong, China

    Lili Zhang & Yan Wang

  2. Applied Oral Sciences and Community Dental Care, Faculty of Dentistry, Prince Philip Dental Hospital, The University of Hong Kong, 34 Hospital Road, Sai Ying Pun, Hong Kong, Hong Kong SAR

    Lili Zhang, Dineshi Sewvandi Thalakiriyawa, Jiawei Liu, Shengyan Yang & Waruna Lakmal Dissanayaka

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Contributions

LZ contributed to the conception, design, data acquisition, analysis, interpretation and drafted the manuscript; DST contributed to data acquisition and analysis, drafted and critically revised the manuscript; JL contributed to data acquisition and analysis; drafted and critically revised the manuscript; SY contributed to the design, analysis, and interpretation, critically revised the manuscript; YW contributed to the design, analysis, and interpretation, critically revised the manuscript; WLD contributed to the conception, design, data analysis, and interpretation, critically revised the manuscript. All authors have read and approved the final manuscript.

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Correspondence to Waruna Lakmal Dissanayaka.

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Ethics approval and consent to participate

This study was performed according to the institutional guidelines. The experimental procedures were carried out in accordance with the protocol entitled “The Hif-1α-SEMA4D-Plexin-B1 signaling axis regulates dental stem cells in the stabilization of vascular structures”, which was initially approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR), The University of Hong Kong on February 21, 2019 (CULATR 4969-19), with an update on July 21, 2021 (CULATR 4969-19 1st Amendment-21). Under this approval, performing experiments using human cells, including applying them in Matrigel plugs under the subcutaneous tissue of mice, was approved. All animal experiments were performed according to the guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health and regulations at the Laboratory Animal Unit of the University of Hong Kong. The work has been reported in line with the ARRIVE guidelines 2.0. The SHED and HUVECs were purchased from AllCells (Alameda, CA, USA) and ScienCell (Carlsbad, CA, USA), respectively. All Cells Co., Ltd and ScienCell Co., Ltd had confirmed in their certificates of analysis (COA) of the product that the ethical approval for collecting human cells was obtained, and the donors had signed informed consent. (AllCells, https://allcells.com/about-allcells/donor-facilities/; ScienCell, https://sciencellonline.com/technical-support/ethical-statement/).

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Zhang, L., Thalakiriyawa, D.S., Liu, J. et al. Semaphorin-4D signaling in recruiting dental stem cells for vascular stabilization. Stem Cell Res Ther 16, 25 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04149-0

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Stem Cell Research & Therapy

ISSN: 1757-6512