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Published in final edited form as: J Invest Dermatol. 2025 Oct 27;146(5):1369–1381.e11. doi: 10.1016/j.jid.2025.10.584

The Role of Endothelial Cell-Derived Extracellular Vesicles in Modulating Fibroblast Function in Skin Wound Healing

Heidi Yuan 1,2, Anna M Salapatas 2, Trevor R Leonardo 2, Chen Han 1,2, Bhavya Vegesna 2, Koushik Debnath 3, Lin Chen 2, Sriram Ravindran 3, Luisa A DiPietro 2
PMCID: PMC13348359  NIHMSID: NIHMS2184982  PMID: 41161638

Abstract

An adequate wound healing response requires the coordination of intercellular signals between multiple cell types. After skin injury, endothelial cells and fibroblasts provisionally replace the site of injury with newly vascularized granulation tissue. Given the spatial and temporal overlap of these 2 cell types in the healing response, we hypothesize that endothelial cell-derived extracellular vesicles (ECEVs) could provide a communication pathway through which endothelial cells influence fibroblast behavior. In this study, we investigated the effects of ECEVs on fibroblast function both in vitro and in vivo through a murine skin wound healing model. Transcriptomic analysis showed that the uptake of ECEVs by fibroblasts altered functions and pathways relating to cell division, extracellular matrix organization, and fibrosis. This gene signature resulted from upregulation of the transcription factor ETV1, which enhanced fibroblast susceptibility to FGF2 found on ECEVs. This transcriptomic analysis was functionally corroborated through in vitro assays in which fibroblasts demonstrated enhanced proliferation, cell cycle progression, and altered extracellular matrix deposition after ECEV treatment. Administration of ECEVs to healing mouse wounds led to increased collagen density and fibroblast quantity in the wound bed of scar tissue. Our study highlights a previously undefined role for ECEVs in regulating fibroblast function during wound repair.

Keywords: Angiogenesis, Exosome, Extracellular matrix, Tissue regeneration, Wound repair

INTRODUCTION

Wound healing is a complex process involving numerous cell types and signaling mechanisms that cooperate to enable proper resolution of a wound (Rodrigues et al, 2019). During the proliferative and remodeling phases, endothelial cells (ECs) and fibroblasts become activated simultaneously to support the formation of healthy, vascularized granulation tissue that provisionally replaces the wounded area (Tefft et al, 2021). Although ECs and fibroblasts engage in the wound healing process in a temporally congruent manner, whether or how intercellular communication occurs between these 2 cell types during wound healing is largely unknown.

Several lines of evidence suggest that the EC–fibroblast interaction may be critical to proper healing. The angiogenic process of healing skin wounds requires an intricate balance between rapid and abundant neovascularization, followed by a regression of the capillary bed to a level of vascularity comparable with that of normal, unwounded tissue (DiPietro, 2016). Although this regulation is fueled in part by GFs induced by the injury response, specific sub-populations of fibroblasts are known to facilitate the growth and retention of this microvasculature (Sorrell et al, 2008). Paracrine interactions between ECs and fibroblasts have been established in 2- and 3-dimensional coculture models of in vitro vasculogenesis (Berthod et al, 2006; Newman et al, 2011; Song et al, 2020; Sorrell et al, 2007; Tefft et al, 2021; Villaschi and Nicosia, 1994), suggesting that fibroblasts support EC functionality at sites of active angiogenesis.

One potential mechanism through which ECs can communicate with fibroblasts and other cell types in the wound is extracellular vesicles (EVs), which are membrane-bound cell-derived particles secreted by nearly all cells (van Niel et al, 2018). EVs can transfer molecules such as proteins, lipids, and nucleic acids to recipient cells (van Niel et al, 2018) in a paracrine, autocrine, or even endocrine manner (Gangoda et al, 2015; Tetta et al, 2013). Although multiple cell types in wounds secrete EVs (Park et al, 2024), EC-derived EVs (ECEVs) are likely a prominent source of wound EVs given the essential role of ECs in angiogenesis (Todorova et al, 2017), endothelial homeostasis (Desideri et al, 2021; Mathiesen et al, 2021), and immune cell modulation (Hosseinkhani et al, 2018; Njock et al, 2015). A plethora of studies have examined the effect of exogenous native or engineered EVs from various multipotent cell types on cutaneous wound healing (Dai et al, 2022; Fernandes et al, 2022; Hu et al, 2016; Huang et al, 2024; Liu et al, 2023a, 2023b; Ren et al, 2022; Wang et al, 2022; Zhang et al, 2022, 2015; Zhou et al, 2022). In contrast, few studies have examined the role of EVs derived from ECs (Fernandes et al, 2022; Liu et al, 2024; Park et al, 2024), let alone their effects on fibroblast function.

In this study, we isolate and characterize EVs from primary human dermal microvascular ECs and demonstrate that they induce transcriptomic and phenotypic changes to dermal fibroblasts in vitro. We further demonstrate that the functional changes associated with fibroblast hyperplasia and ECM deposition are recapitulated through an in vivo murine skin healing model. Our study provides evidence for an understudied communication mechanism in the context of wound repair and potentially other fibrotic pathologies.

RESULTS

Fibroblasts successfully take up ECEVs in vitro

Primary human dermal microvascular ECs were cultured for ECEV isolation as described in the Materials and Methods (Figure 1a). Isolated ECEVs were analyzed using Nanosight Tracking Analysis, transmission electron microscopy, and western blot to confirm their integrity and purity. Nano-particle Tracking Analysis showed that ECEVs exhibited a size range and average consistent with that of small EVs (Welsh et al, 2024) (Figure 1b). In accordance with the Nano-particle Tracking Analysis, transmission electron microscopy demonstrated that ECEVs exhibited a cup-shaped, vesicular morphology within the expected diameter range (Figure 1c). EV markers CD63, CD9, and annexin A2 were also expressed in abundance on both ECEVs and ECs (Figure 1d). β-Actin, an intracellular marker, was absent in the ECEVs, indicating that the ECEV suspension collected is devoid of intracellular components (Figure 1d). Viability of ECs just prior to ECEV collection was high, with negligible levels of cell death (Supplementary Figure S1). To examine fibroblast uptake of ECEVs, fibroblasts were coincubated with green fluorescently labeled ECEVs (Figure 1e) or PKH26 (Figure 1f and Supplementary Figure S2). Two hours after the addition of ECEVs, a dose-dependent and saturable endocytosis of ECEVs by fibroblasts was observed (Figure 1e).

Figure 1. ECEVs from cultured microvascular endothelial cells are endocytosed by dermal fibroblasts.

Figure 1.

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(a) ECEV isolation and resuspension method. (b) Nanoparticle Tracking Analysis of isolated and purified ECEVs. (c) Transmission electron microscopy visualization of ECEV morphology. Yellow bar = 100 nm; white bar = 400 nm. (d) Protein expression of indicated EV markers in isolated ECEVs or ECs. (e) Endocytosis curve of ECEVs: fibroblasts were incubated with increasing concentrations of green fluorescently labeled ECEVs for 2 hours and washed to remove excess ECEVs, and fluorescence intensity was quantified using a microplate reader (left). Fibroblasts were also imaged under a fluorescent microscope to visualize colocalization of labeled ECEVs with intracellular compartments. (f) PKH26-labeled ECEVs were added to fibroblasts, and uptake was visualized through fluorescence microscopy. Upper panel represents matched dye control. Bar = 100 μm (unless otherwise indicated). Data are presented as mean, with data points indicating individual values from quadruplicate cultures. Two independent experiments were performed, and representative experiment is shown. EC, endothelial cell; ECEV, endothelial cell-derived extracellular vesicle; EV, extracellular vesicle.

ECEV uptake shifts the transcriptome of fibroblasts

Next, we examined the transcriptomic changes induced in fibroblasts after exposure to ECEVs. Bulk mRNA sequencing of fibroblasts demonstrated that 2095 genes were differentially expressed after ECEV exposure, with an approximately equal number upregulated (1045) and downregulated (1050) genes (Supplementary Table S1). Samples clustered primarily by treatment group on the basis of principal component and Pearson correlation analyses (Supplementary Figure S3). A gene ontology (GO) analysis of differentially expressed genes (Figure 2a) demonstrated significant upregulation of key biological process terms relating to chromosome segregation, nuclear division, and DNA replication (Figure 2b). Down-regulated GO terms were mainly associated with the extracellular matrix (ECM), such as ECM organization (biological process), extracellular structure organization (biological process), and proteinaceous ECM (cellular component) (Figure 2b). To identify functional pathways affected by ECEV treatment, we performed an analysis with the Reactome database (Milacic et al, 2024) (Figure 2c and Supplementary Table S2). Reactome analysis paralleled the GO term analysis because the top 5 significantly upregulated pathways were associated with fibroblast cell cycle, mitosis, and chromosome activity, whereas the top 5 downregulated pathways were related to ECM organization (Figure 2d).

Figure 2. ECEVs upregulate fibroblast proliferation genes and downregulate ECM-related genes.

Figure 2.

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Confluent fibroblasts were treated with control media or ECEVs for 72 h. Total RNA was extracted from the cells, and bulk mRNA sequencing was performed to identify transcriptomic changes induced by ECEV treatment. (a) All DEGs were identified (adjusted P < .05), and the top 10 MF, CC, and BP terms were ranked on the basis of P-value in descending order. (b) Top 15 upregulated (top) and downregulated (bottom) GO terms ranked from most to least significant in ECEV-treated fibroblasts. (c) Top 15 Reactome pathways in ECEV-treated fibroblasts ranked on the basis of P-value in descending order. (d) Top 5 upregulated (top) and downregulated (bottom) Reactome pathways ranked on the basis of most to least significant in ECEV-treated fibroblasts. (e) IPA was used to identify the top 5 canonical pathways altered by ECEV treatment. Stacked bars refer to percentage of DEGs in ECEV-treated fibroblasts among all genes of each pathway (red = upregulated genes, green = downregulated genes). BP, biological process; CC, cellular component; DEG, differentially expressed gene; ECEV, endothelial cell-derived extracellular vesicle; ECM, extracellular matrix; GO, gene ontology; h, hour; IPA, Ingenuity Pathway Analysis; MF, molecular function.

In addition, we performed a canonical pathway analysis using Ingenuity Pathway Analysis (Qiagen), corroborating the GO and Reactome findings. The top 5 affected pathways involved the cell cycle, ECM organization, and fibrotic signaling (Figure 2e). Pathways predicted to be activated were cell cycle checkpoints, molecular mechanisms of cancer, and kinetochore metaphase signaling pathway. Predicted inhibited pathways were pulmonary fibrosis idiopathic signaling pathway and ECM organization (Supplementary Table S3). In summary, our pathway analysis revealed that ECEVs upregulate genes involved in fibroblast division and replication and downregulate genes related to fibroblast ECM deposition and organization.

ECEVs increase proliferation and cell cycle progression of fibroblasts

To validate the results of the mRNA sequencing analysis, we examined the effect of ECEVs on fibroblast phenotype through a series of functional assays. First, we assessed fibroblast proliferation and progression through the cell cycle. Fibroblasts were seeded at increasing densities, treated with ECEVs for 48 hours, and assessed for proliferation through an MTS assay. Interestingly, even at highly confluent cell densities, ECEV treatment significantly increased fibroblast proliferation (Figure 3a). To understand the cell cycle changes that may underlie this proliferative effect of ECEVs, we performed a cell cycle analysis of ECEV-treated fibroblasts. Flow cytometry demonstrated that ECEV treatment led to a decreased proportion of cells exiting the G1 phase and an increased proportion of cells entering the G2 phase (Figure 3b and c). This observation is consistent with our mRNA-sequencing enrichment analyses, which highlights an upregulation of pathways and gene sets associated with DNA replication and the cell cycle (Figure 2b and d). Taken together, these results show that uptake of ECEVs by human dermal fibroblasts enhances their proliferation and replication.

Figure 3. ECEVs increase proliferation and cell cycle progression of dermal FBs in vitro.

Figure 3.

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(a) FBs seeded at increasing densities up to confluency and subsequently treated with control media (DMEM with 10% exosome-depleted FBS) or ECEVs in control media for 48 h were assessed for proliferation through an MTS assay. (b) FBs were seeded at maximum confluency to induce contact inhibition–mediated cell cycle arrest and then treated with ECEVs for 24 h. Cells were then stained with propidium iodide and analyzed using flow cytometry. Proportion of cells in each phase of the cell cycle was determined using the FlowJo cell cycle analysis function. (c) Representative histogram of stained cells in control (left) or ECEV-treated FB (right) samples identified to be in each phase of the cell cycle using the Watson (pragmatic) curve-fitting algorithm. *P < .05, **P < .01, and ***P < .001. Data are presented as mean ± SD, with data points indicating individual values from triplicate or quadruplicate cultures. Two independent experiments were performed, and representative experiment is shown. Two-way ANOVA with Bonferroni posthoc test was used. ECEV, endothelial cell-derived extracellular vesicle; FB, fibroblast; FBS, fetal bovine serum; h, hour.

ECEVs stimulate the migratory and contractile function of fibroblasts

A critical functionality of fibroblasts in the process of scar formation and wound healing is their ability to migrate across a provisional matrix (Tomasek et al, 2002). We assessed this function using a scratch wound assay. Fibroblasts treated with ECEVs exhibited a greater rate of scratch wound closure within 24 hours of treatment than control fibroblasts (Supplementary Figure S4a), suggesting that ECEVs promote fibroblast migration. A second function we assessed is fibroblast contraction of collagen. Fibroblasts were embedded in a type I collagen gel after treatment with ECEVs, and the surface area of the gel was measured over time as a proxy for contractile capacity (Ngo et al, 2006). ECEV treatment resulted in a smaller gel surface area at each time point assessed, indicating greater contractile function (Supplementary Figure S4b). Altogether, these findings demonstrate that ECEVs enhance the migration and contraction ability of dermal fibroblasts.

ECEVs induce changes in ECM deposition of fibroblasts in vitro

Next, we wanted to evaluate the impact of ECEVs on fibroblast deposition of ECM. To assess this, we seeded fibroblasts at maximum confluency to initiate matrix deposition and treated fibroblasts with ECEVs every 72 hours throughout the course of 14 days. Then, we examined the expression of collagen I and III as well as fibronectin, which is usually highly expressed in mature ECM (Singh et al, 2010). As detected by immunofluorescence, collagen I expression was significantly increased in the matrix deposited by ECEV-treated fibroblasts compared with that of control fibroblasts when normalized to cell nuclei count (Figure 4a). In contrast, fibronectin expression was significantly decreased in response to ECEV treatment (Figure 4b). Collagen III expression was primarily identified to stain intracellularly (Figure 4a) and, therefore, was not significantly affected by ECEV treatment.

Figure 4. ECEVs induce alterations in dermal FB deposition of ECM proteins in vitro.

Figure 4.

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FB were seeded at maximum confluency and then treated with ECEVs every 72 h through day 14; after which, cells and matrix components were fixed and immune stained with antibodies to detect ECM components. (a) Collagen I expression in nondecellularized matrices of FBs treated with control or ECEVs. Total fluorescence area for collagen I and III was quantified with ImageJ and normalized to quantity of nuclei identified by DAPI. (b) Fibronectin and collagen I expression in nondecellularized matrices of FBs treated with control or ECEVs. Total fluorescence area for fibronectin was quantified with ImageJ and normalized to quantity of nuclei identified by DAPI. (c) Day 14 matrices were decellularized, fixed, and stained with picrosirius red. A parallel MTS assay was performed to determine cell density on day 14. Total collagen (red) area was quantified using ImageJ and normalized to a proliferation factor determined by a parallel MTS assay. Bars = 100 μm. *P < .05, **P < .01, and ***P < .001. Mean values for each group are represented by the horizontal lines, with data points indicating individual values obtained from triplicate or quadruplicate cultures. Two independent experiments were performed, and data were combined for a and c, whereas a representative experiment was shown for b. Two-tailed unpaired t-test (vs control) was used. AU, arbitrary unit; ECEV, endothelial cell-derived extracellular vesicle; ECM, extracellular matrix; FB, fibroblast; h, hour.

To visualize and quantify the total collagen deposited by ECEV-treated fibroblasts, matrices were decellularized and stained with picrosirius red. Total collagen, identified by the total red area, was normalized against cellular density. Collagen density was significantly greater in fibroblast-deposited matrices after ECEV treatment (Figure 4c). An analysis of the pattern and organization of the ECM using TWOMBLI (The Workflow of Matrix Biology Informatics) (Wershof et al, 2021), a FIJI macro used for quantifying matrix patterns, showed that collagen matrices derived from ECEV-treated fibroblasts exhibited distinct organization patterns relative to control matrices. In particular, metrics such as high-density matrix, fractal dimension, and endpoint quantity were all increased in ECEV-treated fibroblast ECM (Supplementary Figure S5a). An estimated collagen fiber thickness calculation also revealed that ECEVs promoted the formation of thicker, more dense collagen fibers than control treatment (Supplementary Figure S5a). In conjunction with the immunofluorescence data, these findings reveal that ECEVs can modulate the ability of fibroblasts to deposit collagen and fibronectin in vitro, ultimately influencing the organization and architecture of the ECM.

ECEVs upregulate an FGF2–associated gene signature in fibroblasts mediated by ETV1

To determine the potential mechanism through which ECEVs produced the observed phenotype in fibroblasts, we examined the top 5 upregulated differentially expressed genes in ECEV-treated fibroblasts determined by our RNA-sequencing analysis. We found that the transcription factor ETV1 was among the top 5 upregulated differentially expressed genes (Figure 5a). ETV1 has previously been identified as a critical regulator of dermal fibroblast sensitivity to fibroblast GF 2 (FGF2) and TGF-β1 signaling (Bordignon et al, 2019). Specifically, ETV1 upregulation is associated with increased fibroblast sensitivity to FGF2 signaling, resulting in gene expression changes that correlate with increased cell division and replication and downregulation of ECM organization (Bordignon et al, 2019). To compare the similarity of this FGF2- and ETV1-associated gene signature with that of ECEV-treated fibroblasts, we performed a gene set enrichment analysis of the ECEV-treated fibroblast gene expression profile against the FGF2/ETV1-upregulated genes defined by Bordignon et al (2019) (Supplementary Table S4), This analysis demonstrated significant enrichment of FGF2/ ETV1-associated genes in our dataset (Figure 5b). In addition, we plotted the relative expression of both TGF-β1 and FGF2 effector genes specified by Bordignon et al (2019) and found that ECEV-treated fibroblasts exhibit a dualistic gene expression pattern in favor of FGF2 signaling and against TGF-β1 (Figure 5c).

Figure 5. ECEVs induce an FGF2-associated gene signature mediated by the transcription factor ETV1.

Figure 5.

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(a) Volcano plot showing top 5 upregulated genes found in ECEV-treated fibroblasts derived from RNA-sequencing analysis. (b) GSEA enrichment plot of gene expression profiles of ECEV-treated fibroblasts versus controls against ETV1- and FGF2-associated gene signatures previously defined by Bordignon et al (2019). (c) Gene expression panel showing the downregulation of TGF-β1—associated genes and upregulation of FGF2-associated genes previously defined by Bordignon et al (2019). Log2 fold change values were obtained from RNA-sequencing analysis. (d) FGF2 concentration of unlysed and lysed ECEVs detected by ELISA. (e) FGF2 quantity as detected by ELISA in the conditioned media of fibroblasts treated with control or ECEVs. (f) Protein expression of ETV1 in the whole-cell lysate of fibroblasts treated with control or ECEVs was analyzed using western blot and quantified. *P < .05, **P < .01, and ***P < .001. Data are presented as mean ± SD, with data points indicating individual values from triplicate or quadruplicate cultures. Two independent experiments were performed, and representative experiment is shown. Two-way ANOVA with 2-stage step-up procedure of Benjamini, Krieger, and Yekutieli posthoc test was used. ECEV, endothelial cell-derived extracellular vesicle; FB, fibroblast; FGF2, fibroblast GF 2; GSEA, gene set enrichment analysis.

Given the observed similarity in gene expression patterns between FGF2-upregulated fibroblasts and ECEV-treated fibroblasts, we hypothesized that ECEVs carry active forms of FGF2, either in their intravesicular compartment or tethered to their outer surface, to direct fibroblast behavior. To test this, we assessed relative levels of FGF2 in both lysed and unlysed vesicles and found that ECEVs contain detectable levels of FGF2 within the vesicle and on the membrane surface (Figure 5d). In addition, levels of this GF remained stable in the conditioned media of ECEV-treated fibroblasts through 72 hours of ECEV treatment (Figure 5e), suggesting that ECEVs either increase endogenous production of FGF2 among fibroblasts or potentially stabilize this GF to enhance signal retention and propagation of the ligand to neighboring cells.

Finally, we confirmed that ETV1 expression was also upregulated at the protein level by 48 and 72 hours after ECEV treatment (Figure 5f). Altogether, this implies that the ECEV effect on the fibroblast transcriptome and the associated phenotype involves FGF2 signaling mediated by upregulation of the transcription factor ETV1.

ECEVs induce changes in ECM deposition by fibroblasts in vivo

Given the influence of ECEVs on key fibroblast functions in vitro, we wanted to investigate whether this effect would be recapitulated in an in vivo model of wound healing, which requires a proliferative burst followed by extensive ECM remodeling (Guo and DiPietro, 2010). Mice were wounded on the dorsal skin and treated locally with ECEVs twice weekly (Figure 6a). Wounds were harvested on days 14 and 21 after wounding to examine fibroblast content as well as collagen and ECM deposition. Overall, wound closure was not affected by ECEV treatment (Figure 6b). Trichrome staining revealed that wounds treated with ECEVs had significantly increased collagen in day 14 scars but not in day 21 scars (Figure 6c). However, polarized light microscopy of picrosirius red-stained tissue sections demonstrated an increased ratio of mature to immature collagen in both day 14 and 21 scars (Figure 6d) of ECEV-treated wounds. Fibroblast cell density was significantly increased in day 21 wounds treated with ECEVs (Figure 6e), suggesting that ECEVs augment fibroblast quantity in the wound bed during scar formation. Finally, we assessed fibronectin expression. ECEV treatment had a statistically significant effect on fibronectin deposition (P = .0447) (Figure 6f). Taken together, the findings highlight the role of ECEVs in regulating fibroblast density in the wound bed and the deposition of collagen and ECM.

Figure 6. ECEV treatment of murine wounds increases fibroblast content and alters fibroblast deposition of ECM.

Figure 6.

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(a) Experimental approach: 5-mm fullthickness excisional dorsal wounds on C57/Bl6 wild-type mice were treated with either control vehicle or ECEVs twice weekly and harvested on day 14 (denoted as D14) and 21 (denoted as D21) to assess histology. (b) Wound closure analysis of ECEV-treated or control wounds. Remaining wound area was quantified as a percentage of the original wound size. Data are presented as mean ± SEM, with n = 10 per group at each time point. (c) Cryosections of D14 and D21 control or ECEV-treated wounds were stained with Masson’s Trichrome, and the total area of blue-stained collagen was quantified as a percentage of the analyzed wound bed area. (d) Cryosections of D14 and D21 control or ECEV-treated wounds were stained with picrosirius red and visualized with polarized microscopy. The total area of red/yellow-stained (mature) collagen fibers was quantified as a percentage of the sum of the red/yellow and blue/green-stained (immature) collagen fibers within the wound bed. (e) Cryosections of D14 and D21 control or ECEV-treated wounds were immunostained with antibodies against vimentin. Integrated density was determined by ImageJ and normalized to total wound bed area. (f) Cryosections of D14 and D21 control or ECEV-treated wounds were immunostained with antibodies against fibronectin. Integrated density was determined by ImageJ and normalized to total wound bed area. Bars = 100 μm. *P < .05, **P < .01, and ***P < .001. Data for c–f are presented as mean ± SD, with data points representing wounds obtained from individual mice (n = 5 per group per time point). Two-way ANOVA with 2-stage step-up procedure of Benjamini, Krieger, and Yekutieli posthoc test was used. ECEV, endothelial cell-derived extracellular vesicle; ECM, extracellular matrix.

DISCUSSION

An optimal wound healing response requires the coordination of signals and responses from various cell types (Amiri et al, 2022). In contexts such as tissue injury, this communication likely consists of a combination of both canonical and noncanonical signaling mechanisms. EVs secreted by various resident and infiltrating cells potentially play a large part in the latter because they are implicated in processes such as angiogenesis (Mathiesen et al, 2021; Todorova et al, 2017), macrophage polarization (Zhou et al, 2020), epithelial migration (Zhou et al, 2020), and collagen synthesis (Ding et al, 2023; Zhang et al, 2015), all of which play crucial roles in the functional regeneration of injured tissue. Despite the growing interest in defining the therapeutic potential of EVs derived from multiple cell types, few efforts have focused on those obtained from ECs and their effects on healing and fibrosis. To address this gap, this study focused on ECEVs and did not examine reciprocal activity of fibroblast-derived EVs on ECs. EV-mediated communication between fibroblasts and ECs has been documented in a handful of studies examining fibroblast-derived EVs on EC function in cutaneous wound healing (Bian et al, 2022; Chen et al, 2025; Gangadaran et al, 2020; Oh et al, 2021). These studies found that fibroblast-derived EVs can promote EC activity and angiogenesis both in vitro and in vivo, suggesting that EV-mediated communication between fibroblasts and ECs produces a synergistic effect in favor of healing. In this study, we show that EVs released by dermal microvascular ECs (ECEVs) can induce transcriptomic and phenotypic shifts to dermal fibroblasts and that these alterations affect the properties of the scar tissue resulting from a wound injury.

A predominant phenotype we observed in our analysis of ECEV-treated fibroblasts is increased proliferation. Our findings correlate with those of a previous study, which showed that circulating plasma ECEVs prevented the premature senescence of dermal fibroblasts by promoting YAP nuclear translocation and activating the phosphoinositide 3-kinase/protein kinase B/mTOR signaling pathway (Wei et al, 2020). It is likely that the state or baseline culture conditions or the EC source dictates the resulting properties of the collected EVs. For example, in contrast to our findings, Zeng et al (2019) found that small EVs isolated from human umbilical vein endothelial cells pretreated with advanced glycation end products triggered fibroblast autophagy and suppressed collagen synthesis, ultimately impairing cutaneous wound healing (Zeng et al, 2019). Likewise, EVs collected from apoptotic ECs failed to promote skin fibroblast proliferation in vitro and only weakly promoted migration (Liu et al, 2024). Our study utilized native microvascular ECs that continued to proliferate up until the time of ECEV collection. These ECs would likely be most abundant during the earlier proangiogenic phase of wound repair when new microvasculature is formed, as opposed to the later antiangiogenic phase during which vascular regression and EC apoptosis predominate (DiPietro, 2016). Additional studies could examine the role of EVs derived from ECs cultured in other relevant conditions, including hypoxic or inflammatory states associated with diabetic or chronic nonhealing wounds (Kolluru et al, 2012; Zhao et al, 2016), and their differing effects on fibroblast phenotype.

The hyperplastic, promigratory phenotype of ECEV-treated fibroblasts may initially be unsurprising given the abundance of studies highlighting the enhanced regenerative properties of mesenchymal stem cell-derived EVs in wound repair (Ding et al, 2023; Ren et al, 2019). One striking observation is that the addition of ECEVs seems to abolish the normal contact inhibition that fibroblasts exhibit (Abercrombie, 1970; Pavel et al, 2018), as was observed in the hyperconfluent cell densities used in the MTS assay (Figure 3a) and the ECM deposition assay (Figure 4a and b). A loss of contact inhibition may point to a proinvasive fibroblast phenotype that drives both the overgrowth of cell density and an increase in migratory capacity. Further evidence for the concept that ECEVs can reduce contact inhibition can be found in the gene expression changes induced by ECEVs. The molecular basis of contact inhibition relies on the expression of certain cadherins that mediate cell–cell contact and the regulatory activity of small GTPases, specifically the Rho family of GTPases (Roycroft and Mayor, 2016). Our analysis showed that integrin cell surface interactions were downregulated (Supplementary Table S2) and that the gene for N-cadherin (CDH2) in fibroblasts was also suppressed after ECEV treatment (Supplementary Table S1). In addition, the Reactome pathway Rho GTPases activate formins was dysregulated (Figure 2c and Supplementary Table S2), suggesting that Rho-dependent cytoskeletal rearrangements implicated in contact inhibition (Hall and Nobes, 2000) could be impaired.

Our transcriptomic data also provide insight into the potential signaling mechanisms that underlie the proliferative effects of ECEVs on fibroblasts. Overall, fibroblasts treated with ECEVs exhibited gene expression changes that promoted cell division and mitosis and downregulated genes coding for ECM-related molecules (Figure 2). This distinct gene expression pattern, controlled by the transcription factor ETV1, enabled us to identify the potential contribution of FGF2 in the ECEV-induced fibroblast effect (Figure 5). As first reported by Bordignon et al (2019), cancer-associated fibroblasts expressing high levels of ETV1 are predisposed to FGF2 signaling while simultaneously downregulating canonical TGF-β1 signaling. The resulting cancer-associated fibroblast phenotype was one that increased inflammation and macrophage recruitment, ultimately eliciting a protumorigenic microenvironment. This shared gene expression pattern between cancer-associated fibroblasts and ECEV-treated fibroblasts provides evidence to implicate ECEVs in the formation of tumor stromal tissue or cancer-associated fibrosis, given the links between angiogenesis and tumor growth (Nishida et al, 2006).

Our data showed that ECEV-treated fibroblasts similarly upregulate ETV1 expression at the gene and protein levels (Figure 5a and f). We also found that ECEVs contain FGF2 likely on the cell surface and within their intravesicular compartment (Figure 5d), and levels of this GF remained increased in the conditioned media of treated fibroblasts compared with that of controls (Figure 5e). Given these findings, we hypothesize that ECEVs could influence fibroblast sensitivity to FGF2 signaling in several ways (Figure 7). First, they upregulate ETV1. Second, ECEVs may contain heparan sulfate proteoglycans on their surface (Cerezo-Maganã et al, 2020), acting as a crucial reservoir of this essential cofactor to enhance FGF2 secretion and stability in the extracellular space (Shimokawa et al, 2011; Zehe et al, 2006). Third, ECEV-stimulated fibroblasts can further release their own FGF2-bound EVs (Petit et al, 2022; Taverna et al, 2003), thus facilitating a positive feedback loop in which endogenous FGF2 ligands are secreted and propagated to neighboring cells. Future directions of this research should examine the precise mechanisms of vesicle-bound FGF2 on recipient cell dynamics.

Figure 7. Proposed mechanism of action of ECEVs on fibroblasts involving FGF2 and the transcription factor ETV1.

Figure 7.

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ECEVs released by endothelial cells contain FGF2, both in their intravesicular compartment and on the outer surface. These FGF2 ligands are likely tethered to HSPGs, which can stabilize the GF and act as a cofactor for signal induction. Uptake of ECEVs by fibroblasts leads to upregulation of ETV1, predisposing fibroblasts to FGF2 signaling and upregulating cell division genes and downregulating ECM genes. FGF2 signal propagation among fibroblasts can further occur through 2 possible mechanisms: either through endogenous secretion of FGF2 to neighboring fibroblasts or through fibroblast release of EVs containing FGF2. ECEV, endothelial cell-derived extracellular vesicle; ECM, extracellular matrix; EV, extracellular vesicle; FGF2, fibroblast GF 2; FGFR, fibroblast GF receptor; HSPG, heparan sulfate proteoglycan.

In wounds, FGF2 has long been recognized as crucial for repair, possessing mitogenic properties, enhancing cell survival, and promoting angiogenesis (Farooq et al, 2021). To our surprise, we were unable to detect a significant change in vascularity, as measured by CD31 density in day 14 and day 21 scars, in ECEV-treated wounds (Supplementary Figure S6a and b). This could be due to the strongly antiangiogenic signals present in the wound at the later healing phase to promote vascular regression (DiPietro, 2016; Wietecha et al, 2011). Previous work in our laboratory showed that administering exogenous proangiogenic GFs well above physiological levels does not inhibit vascular regression in late-stage wounds (Gosain et al, 2006). This suggests that the antiangiogenic stimuli present in wounds may override any proangiogenic effects of ECEVs or their associated GFs.

Although angiogenesis was not significantly affected at the measured time points, fibroblast activity and ECM deposition showed apparent changes in response to ECEVs. ECEVs altered the architecture of fibroblast-deposited collagen and fibronectin matrices (Figure 4a and b). Collagen matrices deposited by ECEV-treated fibroblasts exhibited greater density with thicker collagen fibers, as analyzed by TWOMBLI (Wershof et al, 2021) (Supplementary Figure S5a). This aligned with the increased total collagen deposition observed in day 14 mouse wounds treated with ECEVs (Figure 6c) and the upregulated mature type I collagen density in both day 14 and day 21 wounds (Figure 6d). Similarly, we analyzed both fibronectin deposition (Figures 4b and 6f) and organization (Supplementary Figure S5b). Fibronectin quantity was decreased in cell-derived matrices and wounds treated with ECEVs, and its organization was markedly affected. Fibronectin plays a crucial role in scaffolding the ECM, and its polymerization into fibrils regulates ECM stability and the adhesion between cells and the matrix (Sottile and Hocking, 2002). It also has an established role in organizing the initial processing of procollagen to support proper collagen fibrillogenesis (Saunders and Schwarzbauer, 2019). The observed imbalance of low fibronectin and high type I collagen density in the resulting ECM suggests that ECEVs may disturb the optimal dynamics required for an intact, stable collagen network and instead subtly predispose the wounds toward a profibrotic, scarring phenotype. Taken together, the documented changes to fibroblast functionality suggest a potentially disadvantageous role for ECEVs in fibroblast activation, especially in contexts where aberrant hypervascularity occurs, such as tumor growth or cancer. Our findings further underscore the importance of refined angiogenesis as a critical regulator of fibrotic outcomes in wounds.

It is also noteworthy to mention that although some ECM-related genes, including the genes coding for collagen I (Figure 5c), were downregulated, our in vitro and in vivo analyses demonstrated an increase in the protein expression of collagen I, even when proliferation was accounted for (Figures 4a-c and 6c and d). Although this gene and protein mismatch at first appears confounding, it may be explained by the experimental setup. Fibroblasts were grown over the course of 14 days, which was a necessary step to provide sufficient time for matrix maturation (Murphy et al, 2022). However, this timeline would likely have permitted the assembly of collagens by a few generations of fibroblasts, which may not have been accurately captured by the cell density or viability we measured at the time of assessment. It is therefore imperative to recognize that intracellular mRNA levels cannot always fully predict the resulting composition and dynamics of the ECM (Naba, 2023). Instead, the protein expression and matrix analysis likely provide the most accurate representation of ECEV effect on fibroblast ECM.

In conclusion, our findings indicate a prominent role for EVs in facilitating intercellular communication between ECs and fibroblasts in the context of wound healing. We found transcriptomic shifts that correlate with a pro-proliferative, ECM remodeling phenotype in fibroblasts that is consistent with a previously reported ETV1-mediated gene signature in cancer-associated fibroblasts. Importantly, treatment of wounds with ECEVs resulted in altered collagen synthesis and ECM dysregulation within scar tissue. Overall, our work highlights ECEVs as an important regulator of fibroblast behavior and provides evidence for their potential involvement in the relationship between angiogenesis and fibrosis.

MATERIALS AND METHODS

Full experimental details are provided in Supplementary Materials and Methods.

Cell culture and ECEV isolation

Primary human dermal microvascular ECs (ATCC, #FC-0042) were cultured in vascular cell basal media supplemented with GFs and 5% fetal bovine serum. At confluence, cells were washed and incubated in exosome-free media (vascular cell basal media with GFs and 5% exosome-depleted fetal bovine serum). After 48 hours, conditioned media were sequentially centrifuged (300g, 5 minutes; 3000g, 15 minutes) to remove debris. ECEVs were precipitated using a 4× polyethylene glycol–based isolation reagent, incubated over-night at 4 °C, and pelleted (1500g, 30 minutes). Pellets were resuspended in PBS, normalized to originating cell number (1 μl per 1 × 104 cells).

Validation of ECEVs

Size and concentration were determined by Nanoparticle Tracking Analysis (NanoSight NS300). Morphology was visualized by transmission electron microscopy. EV markers (CD63, CD9, CD81, and annexin A2) and β-actin were analyzed by western blot.

ECEV uptake by fibroblasts

ECEVs were fluorescently labeled (CellTracker Green CMFDA or PKH26) and incubated with primary human dermal fibroblasts (Gibco, #C0045C). Uptake was quantified by a fluorescence plate reader and confirmed by microscopy.

Fibroblast functional assays (proliferation, cell cycle, migration, and collagen contraction)

Human dermal fibroblasts were treated with 30% v/v ECEVs or control medium (10% exosome-depleted fetal bovine serum in DMEM). Proliferation was assessed by MTS assay, cell cycle was assessed by flow cytometry, migration was assessed by scratch assay, and contractility was assessed by collagen gel contraction assay.

ECM deposition assay

Human dermal fibroblasts were cultured with ECEVs or control medium for 14 days. Collagen I, collagen III, and fibronectin deposition were evaluated by immunofluorescence. Collagen content was further quantified by picrosirius red staining after decellularization and normalized to proliferation, and collagen architecture was analyzed using TWOMBLI.

FGF2 and ETV1 protein analysis

FGF2 in ECEVs and fibroblast-conditioned media was quantified by ELISA. ETV1 expression in fibroblasts was analyzed by western blot.

RNA sequencing

Human dermal fibroblasts were treated with ECEVs or control medium for 72 hours. Total RNA was extracted and sequenced (Illumina). Reads were aligned with Hisat2, assembled with StringTie, and analyzed for differential expression with DESeq2. GO and pathway enrichment analysis were performed using GO, Reactome, Ingenuity Pathway Analysis, and Gene Set Enrichment Analysis.

In vivo wound healing analysis

Full-thickness excisional wounds (5 mm in diameter) were generated on the dorsum of C57BL/6 mice. Wounds were treated with 1 × 109 ECEVs or vehicle (5% DMSO in PBS) twice weekly. Wound closure was monitored by digital photography, and wound tissues were collected at days 14 and 21 after wounding for histological analysis.

Histology and immunofluorescence

Wound cryosections were stained with Masson’s trichrome and picrosirius red to assess collagen content and maturity. Immunofluorescence staining was used to identify fibroblasts (vimentin), fibronectin, and vessel density (CD31). Images were quantified with ImageJ.

Statistical analysis

Data are shown as mean ± SD unless otherwise specified. Comparisons were made using unpaired t-tests or 2-way ANOVA with post-hoc tests (GraphPad Prism, version 10.3.0). Significance was set at P < .05.

Supplementary Material

1
2

Supplementary material is linked to the online version of the paper at www.jidonline.org, and at 10.1016/j.jid.2025.10.584.

ACKNOWLEDGMENTS

We would like to thank Mateusz Wietecha for his guidance on the analysis of the RNA-sequencing data and Wendy L. Cerny and Yixuan Zhang for editing and providing feedback on this manuscript. This research was funded by National Institutes of Health R01-GM50875 and R35-GM139603 (for LAD), F31-AR083830 (for HY), F30-DE029689 (for AMS), F31-DE028747 (for TRL), F31-AR082287 (for CH), and R01-DE027404 and DE30495 (for SR). The abstract has previously been presented at the Society for Investigative Dermatology 2025 meeting and published in Journal of Investigative Dermatology.

Abbreviations

EC

endothelial cell

ECEV

endothelial cell-derived extracellular vesicle

ECM

extracellular matrix

EV

extracellular vesicle

FGF2

fibroblast GF 2

GO

gene ontology

Footnotes

ETHICS STATEMENT

This study was performed in accordance with the Declaration of Helsinki. Human primary cell lines included in this study were approved as part of this study protocol. Ethical approval was not received for this human study because all primary cells were purchased from Lifeline Cell Technology, which states that all donated tissues have been obtained under written informed consent and adheres to the Declaration of Helsinki, The Human Tissue Act (United Kingdom), CFR Title 21, and Health Insurance Portability and Accountability Act Regulations relative to obtaining and handling human tissue for research use. Collection of animal tissue samples for this study was approved as part of the study protocol. This animal study was approved by University of Illinois Chicago Research Institutional Animal Care and Use Committee (approval 2023-155). All animal procedures were carried out in accordance with protocols approved by the University of Illinois Chicago Institutional Animal Care and Use Committee.

CONFLICT OF INTEREST

The authors state no conflict of interest.

DATA AVAILABILITY STATEMENT

Datasets related to this article can be found in National Center for Biotechnology Information Gene Expression Omnibus with accession number GSE293186. Primary data related to this publication can be accessed via INDIGO with the DOI: 10.25417/uic.30592964.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS2184982-supplement-1.xlsx (328.4KB, xlsx)
2
NIHMS2184982-supplement-2.pdf (25.9MB, pdf)

Data Availability Statement

Datasets related to this article can be found in National Center for Biotechnology Information Gene Expression Omnibus with accession number GSE293186. Primary data related to this publication can be accessed via INDIGO with the DOI: 10.25417/uic.30592964.

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