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19 February 2026

17 Pages

Plasma Extracellular Vesicles from Bronchopulmonary Dysplasia Infants Initiate Inflammation and Abnormal Angiogenesis in Neonatal Murine Retinas

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Division of Neonatology, Department of Pediatrics, Batchelor Children’s Research Institute, Miller School of Medicine, University of Miami, Miami, FL 33136, USA
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Bascom Palmer Eye Institute, Miller School of Medicine, University of Miami, Miami, FL33136, USA
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Byers Eye Institute, Stanford University, Palo Alto, CA 94305, USA
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Author to whom correspondence should be addressed.
Cells2026, 15(4), 367;https://doi.org/10.3390/cells15040367
This article belongs to the Section Cell Microenvironment
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Highlights

What are the main findings?
  • Adoptive transfer of plasma extracellular vesicles from bronchopulmonary dysplasia infants (BPD-EVs) into neonatal mice on postnatal day 3 (P3) induced activated retinal microglia, Müller cells, and abnormal neovascularization on P17 compared to nBPD-EVs.
  • Proteomics analysis of BPD-EVs and nBPD-EVs revealed that BPD-EVs had significantly elevated levels of inflammation and angiogenesis-related proteins compared to nBPD-EVs.
What are the implications of the main findings?
  • Circulating EVs may contribute to retinopathy of prematurity (ROP) development in BPD infants by inducing retinal inflammasome-associated inflammation and abnormal angiogenesis.
  • Inflammation and angiogenesis-related proteins in BPD-EVs may represent novel biomarkers and therapeutic targets for the treatment of ROP.

Abstract

Purpose: To investigate the mechanisms by which plasma extracellular vesicles (EVs) from preterm infants with bronchopulmonary dysplasia (BPD) elicit inflammation and abnormal angiogenesis in neonatal mouse retinas. Methods: EVs from the plasma of 7-day-old preterm infants, born between 230/7 and 296/7 weeks of gestation, with BPD or without BPD (nBPD) at 36 weeks postmenstrual ages, were adoptively transferred into postnatal day 3 (P3) mice via intravenous retro-orbital sinus injection. Inflammation and pathological neovascularization in neonatal mouse retinas were examined by immunohistochemistry of retinal flat mounts for Allograft Inflammatory Factor 1 (AIF1), CD206, or Glial Fibrillary Acidic Protein (GFAP) and isolectin-B4 (IB4) staining on P17. Retinal inflammation-related transcripts were assessed by qRT-PCR. Proteomic profiles of BPD and nBPD EVs were examined by Liquid Chromatograph Mass Spectrometer/Mass Spectrometer (LC-MS/MS) and Gene Set Enrichment Analysis (GSEA). Results: Adoptively transferred EVs from BPD and nBPD infants crossed the blood–retinal barrier (BRB) in recipient mouse pups. BPD-EVs increased retinal activated microglia, Müller cells, and twisted proliferative neovascularization compared to nBPD-EVs. BPD-EVs also elevated retinal transcripts regulating inflammation and angiogenesis, including NOD-, LRR- and pyrin domain-containing protein 3 (Nlrp3), Apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc), Caspase 3 (Casp3), Caspase 8 (Casp8), Gasdermin D (Gsdmd), Il1β, Il6, Aif1, and Vascular endothelial growth factor (Vegf). Proteomics analysis revealed that BPD-EVs had significantly elevated levels of inflammation and angiogenesis-related proteins compared to nBPD-EVs. Conclusions: BPD-EVs promote inflammation and abnormal neovascularization by upregulating genes related to apoptosis and inflammation in neonatal mouse retinas. EV protein profiles suggest that elevated levels of proteins such as Defensin alpha 1B (DEFA1B), Insulin-like growth factor binding protein 2 (IGFBP2), CD5 antigen-like (CD5L), von Willebrand factor (vWF), and Tenascin C (TNC) in BPD-EVs may contribute to the observed inflammation and angiogenesis.

1. Introduction

Bronchopulmonary dysplasia (BPD) is a chronic lung disease in infants born extremely prematurely with very low birth weights who undergo oxygen treatment. Hyperoxia-induced inflammatory injuries with short- and long-term consequences have been documented in multiple developing organs, including the eyes (ROP), lungs (BPD), and brains (NDI: neurodevelopment impairment) [1,2,3]. Retinopathy of prematurity (ROP) is common in very-low-weight preterm infants, many of whom require oxygen therapy due to lung underdevelopment [1,4]. The mouse oxygen-induced retinopathy (OIR) model has been widely used as a paradigm for human ROP. In this model, oxygen-induced vascular disorders occur primarily in the central retina with two disease phases: an initial vaso-obliteration phase with oxygen exposure from postnatal day 5 (P5) to P12, resulting in retinal hypoxia, and a subsequent abnormal twisted proliferative neovascularization phase peaking on P17 [5]. Phase II is characterized not only by abnormal angiogenesis induced by elevated VEGF but also by a strong inflammatory response by the activation of microglia and Müller cells [6]. Pathological angiogenesis, a hallmark of severe ROP, is induced primarily by aberrant regulation of angiogenic factors signaling under the control of inflammatory mediators [7,8]. However, the etiological mechanisms of hyperoxia-induced retinal injury remain to be elucidated, and further studies are essential to fully understand these mechanisms and identify methods to predict, prevent, or treat ROP.
Extracellular vesicles (EVs) are cell-derived membranous structures of 30 to 200 nm in size that regulate intercellular and interorgan communication [9]. EVs are present in various biological fluids, including blood, where their concentrations have been observed to increase during inflammatory responses in diseases such as cancer, HIV infection, and inflammatory disorders [10,11,12]. This phenomenon has been extensively studied in the context of multiple cancers and inflammatory diseases, highlighting its potential as a disease mediator, biomarker, and therapeutic target [13,14,15]. Moreover, EVs, including those from the blood of BPD patients and rodent models of BPD, have been identified as novel mediators of BPD-associated lung and brain injury [16,17]. Our previous studies suggest an activated inflammasome pathway may contribute to BPD development, and we recently found that circulating EVs derived from the plasma of BPD patients contain elevated cargos of the inflammasome adaptor, ASC, and inflammation-related cytokines [16,18]. The adoptive transfer of these circulating BPD-EVs into neonatal mice induced inflammation in the lungs and hippocampus, accompanied by reduced cell survival [16]. In addition, our recent investigations have also shown that ASC expression was markedly enhanced in the retinas of wild-type or ASC reporter mice with oxygen-induced retinopathy (OIR) and that ASC specks colocalized with microglia and abnormal angiogenesis in these retinas [19]. However, studies directly linking circulating BPD-EVs to the induction of inflammation and abnormal angiogenesis in neonatal mouse retinas have not yet been conducted.
Thus, in this study, we adoptively transferred plasma EV from infants with BPD or without BPD (nBPD) into postnatal day 3 (P3) mouse neonates and showed that the retinas of mice injected with BPD-EVs on P17 had increased retinal microglia and Müller cell numbers, abnormal retinal angiogenesis and increased retinal RNA transcripts associated with apoptosis, inflammasome-mediated inflammation and pyroptosis with related cytokine expression. Additionally, when the protein profiles of these EVs were assayed by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) and analyzed using Gene Set Enrichment Analysis (GSEA), we found significant alterations in proteins related to inflammatory responses and abnormal pathological angiogenesis in BPD-EVs compared to nBPD-EVs.

2. Materials and Methods

2.1. Human Study Design and Blood Sample Collection

This study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Boards of Jackson Health System and the University of Miami Miller School of Medicine (protocol #20200736/18 June 2020). This prospective, longitudinal, and observational study was conducted in the neonatal ICU (NICU) at the Holtz Children’s Hospital in the Jackson Health System and the University of Miami Miller School of Medicine. The study population consisted of neonates with gestational age (GA) 230/7–296/7 weeks admitted to our NICU between August 2021 and August 2022. Written informed parental consent was obtained at <7 days of age upon admission to the NICU. The patients’ demographic characteristics were prospectively collected and entered into the database as previously described [16]. BPD was defined using the 2001 Eunice Kennedy Shriver National Institute of Child Health and Human Development workshop [16]. Blood specimens were obtained within 7 days of life, and plasma was collected in EDTA tubes and stored at −80 °C for future analyses [16].

2.2. Animals and Study Approval

All animal procedures complied with ARRIVE guidelines (Animal Research: Reporting In Vivo Experiments), and the study (protocol #23-039/8 April 2023) was approved by the Animal Care and Use Committee of the University of Miami Miller School of Medicine. Wild-type mice (C57BL/6J) were obtained from the Jackson Laboratory, Bar Harbor, ME, USA. All neonatal mice were bred from C57BL/6J wild-type mice. Neonatal mice were housed with mixed littermates in standard individually ventilated cages under specific pathogen-free conditions. Mice were maintained at 20–24 °C with 40–60% relative humidity on a 12:12 h light–dark cycle. Pups were nourished via mothers. Cages were provided with bedding and nesting material to support maternal behavior and pup thermoregulation. Cage changes and handling were minimized during the neonatal period.

2.3. EV Isolation, Labeling, and In Vivo Tracking

Plasma EVs were isolated, identified, and characterized as we have previously described [16]. We randomly selected seven plasma samples from infants with BPD, seven from infants without BPD, and one adult human plasma sample (Sigma-Aldrich, St. Louis, MO, USA), and we isolated EVs from these samples using ExoQuick (System Biosciences, Palo Alto, CA, USA, No. EXOQ5A-1) according to the manufacturer’s instructions. Briefly, after thrombin treatment to digest fibrin, 100 µL of plasma sample was centrifuged at 3000× g for 10 min to eliminate cells and cell debris. The supernatant was collected and mixed with an appropriate volume of precipitation solution in an Eppendorf tube for 30 min at 4 °C. The mixes were centrifuged at 1500× g for 30 min, and the EV pellets were resuspended in 100 µL of PBS. Protein concentration in each EV preparation was measured by BCA protein assay using a commercial kit from Pierce Biotechnology, Rockford, IL, USA. These EVs meet the MISEV2023 guidelines for EVs [20], as nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) show that they are intact and have a diameter of 100–150 nm, while western blot analysis indicates they have EV membrane markers CD9, CD63, and CD81 [16]. Plasma-derived EVs are inherently co-isolated with some abundant plasma proteins. This limitation is explicitly acknowledged in the MISEV2023 guidelines and applies to all plasma EV studies, regardless of isolation method [20].
To assess if circulating EVs can cross the blood–retinal barrier (BRB), EVs were labeled with an ExoGlow-Vivo EV near-infrared Labeling Kit (SBI, Cat. EXOGV900A-1) or an ExoGlow EV Cargo-Protein green, fluorescent Labeling Kit (Cat. EXOGP300A-1), according to the manufacturer’s instructions (System Biosciences, Palo Alto, CA, USA). Several studies have shown that EVs labeled with these methods (non-lipophilic dyes) are taken up in vivo and in vitro with high sensitivity and specificity while maintaining EV integrity [21,22]. Labeled BPD-EVs, nBPD-EVs, or sham-labeled PBS were adoptively transferred into P10 neonatal mice via intravenous retro-orbital sinus injection. Mouse organs were dissected 2 h after the administration of EV injections [23]. IVIS or confocal microscopy images were acquired to analyze EV distribution in tissues or retinas.

2.4. Adoptive Transfer of EVs by Intravenous Retro-Orbital Sinus Injection

The mouse pups were randomly assigned to three groups derived from mixed litters after birth. In our preliminary study, we found no significant effect of sex on the inflammatory response in neonatal mice. A total of 15 micrograms/10 µL of pooled EVs from 7 BPD infants or 7 nBPD infants, as well as EVs from pooled adult human plasma (Sigma-Aldrich, St. Louis, MO, USA), were administered to P3 neonatal mouse pups via retro-orbital sinus injection (n = 5 mice/group). The experiment was repeated 3 times (total: n = 10–15 mice/group). Normal adult human EVs (Hu-EVs) served as negative EV controls versus neonatal nBPD-EVs, as these non-neonatal EVs had no effect on ROP induction in preliminary assays, similar to what we have previously reported for PBS [19]. Pups, with an average weight of 7 gm, were euthanized on P17, and one eye from each animal was enucleated and fixed in 4% paraformaldehyde for 45 min at room temperature for retinal whole-mount analysis. The other eye was used for retinal dissection and subsequently stored at −80 °C for RNA analysis [24].

2.4.1. Analysis of Retinal Whole-Mount Pathology with Immunofluorescent Staining

Retinas were dissected and stained as previously described [19]. Briefly, after blocking with 1% donkey serum in PBS, the retinal whole mounts were stained with AF-594-labeled isolectin B4 (IB4) for retinal vessel labeling, and with goat anti-allograft inflammatory factor 1 (AIF1) IgG for microglial staining, rabbit anti-glial fibrillary acidic protein (GFAP) IgG for Müller cells or rat AF-647-labeled IgG for anti-CD206, a marker for activated microglial cells, all overnight at 4 °C. AIF1 and GFAP IgG were detected by anti-goat or -rabbit IgG labeled with AF-488. All immunohistochemistry procedures were performed using standardized methods with the same antibody concentration across all treatment groups and experiments. After flat mounting, retinal images were acquired using the same imaging settings across all groups, and the abnormal proliferative neovascular area was analyzed using fluorescent microscopy (Zeiss software, ZEN 3.1 Blue Edition, Carl Zeiss Microscopy, White Plains, NY, USA). Five or ten randomly selected, non-overlapping photomicrographic images of each flat mount were also taken for AIF-1, GFAP or CD206 co-staining with IB4 and blindly analyzed by imageJ software (v1.51a, NIH, Bethesda, MD, USA) and presented as the mean ± SEM. Additional antibody information is included in Supplementary Table S1.

2.4.2. RNA Isolation and Quantitative RT-PCR

Total RNA was extracted from retinas using Trizol (Qiagen Science, MD, USA), followed by cDNA synthesis utilizing the iScript cDNA synthesis Kit. Gene expression levels for Casp3, Casp8, Asc, Nlrp3, Gsdmd, Vegf, Aif1, Il1b and Il6 were quantified by qRT-PCR with Taqman gene expression assays using a Bio-Rad Laboratories (Hercules, CA, USA) cycler system. Primers for these genes were sourced from Thermo-Fisher Scientific, Waltham, MA, USA. The relative gene expression levels were normalized to 18S RNA. The details of all reagents and primers are described in Supplementary Table S1.

2.5. Human Retinal Microvascular Endothelial Cell Culture and TUNEL Assay

Human retinal microvascular endothelial cells (HRMEC, Cat. No. ACBRI 181, Cell Systems, Kirkland, WA, USA) were cultured according to manufacturers’ instructions. A total of 1.2 × 104 cells per well were seeded on cover slips in 48 well plates with or without 10 µg of EVs per well in triplicate at 37 °C for 48 h. A TUNEL assay (Thermo-Fisher Scientific) was performed following the manufacturer’s instructions to determine cell death using DNA fragmentation as previously described [17].

2.6. EVs Mass Spectrometry Analysis

The proteomic profiles of plasma EVs were analyzed by LC-MS/MS at the Harvard Medical School Mass Spectrometry Facility. Briefly, 40 µg of EVs were digested with trypsin, and the resulting peptides were separated, detected, isolated, and fragmented to generate a tandem mass spectrum of specific fragment ions for each peptide. Peptide sequences and protein identities were determined by matching the acquired fragmentation patterns with protein databases using the Sequent software program (Thermo-Fisher Scientific, Waltham, MA, USA). All databases included a reversed version of all sequences, and the data were filtered to achieve a nearly 1% peptide false discovery rate (FDR). The mass spectrometry proteomics data have been deposited into ProteomeXchange via the PRIDE partner repository dataset (Public access code: PXD068467).

2.7. Bioinformatic Analysis

Proteomic group comparisons were analyzed by GSEA using the Benjamini–Hochberg method to identify significant data with functional interpretation of the proteomic data with p-values adjusted by an FDR calculator (https://tools.carbocation.com (accessed on 31 May 2024)). The plots were performed using SRPLOT online software (http://www.bioinformatics.com.cn/SRplot (accessed on 8 June 2024)) for functional interpretation of the proteomic data with p-values adjusted by the FDR.

2.8. Statistics

Statistical comparisons among EV-treated groups were performed using ANOVA followed by Tukey’s post hoc test utilizing Prism v9.0 software (GraphPad Software Inc., San Diego, CA, USA). Data are expressed as mean ± SEM, with statistical significance set at p < 0.05.

3. Results

3.1. Adoptively Transferred BPD-EVs and nBPD-EVs Cross the BRB in Neonatal Mouse Retinas

Our previous studies show that BPD-EVs injected into neonatal mice are taken up not only by the lungs but also can cross the blood–brain barrier (BBB) and be taken up by brain cells [16]. To determine if BPD- and nBPD-EVs can also cross the BRB and be taken up by eyes, we assessed the ability of labeled EVs to be taken up by eyes and lungs after retro-orbital intravenous injection in mice at P10, as illustrated in the upper panel of Figure 1I. The biodistribution of EVs was tracked using IVIS, which revealed robust signals from EVs labeled with ExoGlow™-Vivo. The EVs were detected in the lungs (Figure 1II(B,C)) and eyes (Figure 1II(E,F)) 2 h after injection for both BPD- and nBPD-EVs, while no signals were observed in the PBS controls (Figure 1II(A,D)). Similarly, immunofluorescent confocal microscopy images showed the presence of ExoGlow™ fluorescent-green labeled BPD- and nBPD-EVs 2 h after injection in mouse retinal whole mounts co-stained with IB4 (Figure 1II(H,I,K,L,N,O)) but were absent in PBS controls (Figure 1II(G,J,M)). These results suggest that EVs can effectively cross the BRB into mouse retinas following intravenous injections. Moreover, BPD-EVs accumulated more in lungs and eyes than did nBPD-EVs, suggesting that BPD-EVs are preferentially taken up by BPD- or ROP-associated organs, as we previously have shown for lung and brain [16]. Similar organ-specific uptake has been reported for organs afflicted by other diseases [19,23].
Figure 1. Biodistribution of labeled EVs. (I). Study protocol for biodistribution of labeled EVs following retro-orbital sinus intravenous injection (Upper panel; created in https://BioRender.com). (II). Assessment of retinal EV uptake (Bottom panel). (II(A–F)). Representative IVIS images of ExoGlow-Vivo-labeled PBS, nBPD-EVs, or BPD-EVs in the lungs (II(A–C)) and eyes (II(D–F)) at 2 h post EV injection. (II(G–O)). Representative fluorescent confocal microscopy 3D images of ExoGlow-Green-labeled EVs in mouse retinas. (II(G,J,M)). Sham-labeled PBS-injected control. (II(H,K,N)). nBPD-EV injected. (II(I,L,O)). BPD-EV injected. (II(G–I)). Green: ExoGlow-green-labeled EVs. (II(J–L)). Red: IB4-AF594 staining of retinal blood vessels. (II(M–O)). Merged images of green-labeled EVs and red IB4-stained blood vessels (Scale bars: 70 µm).

3.2. Adoptively Transferred BPD-EVs Induce Inflammatory Responses and Pathological Angiogenesis in Neonatal Mouse Retinas

To investigate whether BPD-EVs contribute to neonatal mouse retinal inflammation and pathological angiogenesis, a total of 15 µg of EVs in 10 µL PBS pooled from the plasma of 7 infants with BPD or nBPD and adult humans, respectively, was administered to neonatal mice by retro-orbital intravenous injection on P3. Subsequently, we assessed retinal whole-mount vascularization and inflammatory cell infiltration after sacrifice on P17 using immunofluorescent confocal microscopy as illustrated in the upper panel of Figure 2I. IB4-stained retinal whole mounts revealed around 20% pathological twisted proliferative neovascularization in the retinas of BPD-EV injected mice (Figure 2II(C,D)), compared to only 2–5% in the retinas of nBPD-EV- and human-EV-injected mice (Figure 2II(A,B,D)). Double immunostaining with anti-AIF1 and IB4 indicated that the pathological twisted proliferative neovascularization was colocalized with activated AIF1 positive microglial cells (Figure 3C). The intensity of neovascularization as well as the number of microglial cells was significantly increased approximately twofold greater in the retinas of mice injected with BPD-EV compared to those injected with nBPD-EVs and Hu-EV controls (Figure S1D,E). Immunostaining with anti-GFAP or anti-CD206 and IB4 revealed a significant increase of greater than twofold in Müller cells (GFAP+) and activated microglia cells (CD206+) (Figure 4C,I,M,N), both of which were colocalized with abnormal proliferative neovascularization within the retinas of BPD-EV-injected mice compared to nBPD-EV- and human-EV-injected controls (Figure 4J,K,L).
Figure 2. BPD-EVs promote pathological angiogenesis and inflammation. (I). Study protocol for EV adoptive transfer and retinal analyses (Upper panel). (II). BPD-EVs promote pathological angiogenesis. (II(A–C)). Representative IB4-AF594 (Red) stained total retina images of P17 neonatal mouse retinal vasculature following P3 adoptive transfer of Hu-EVs (II(A)), nBPD-EVs (II(B)) and BPD-EVs (II(C)). (White areas indicate the areas of twisted proliferative neovascularization.) Scar bar: 50 µm. (II(D)) Quantification of the percentage of neovascularization (n = 10–15 retinas/group, **** p < 0.0001, ns: no significance). Scale bar: 50 µm.
Figure 3. BPD-EVs increase microglial cell number in the retinas of neonatal mice. (A–C). Images of merged anti-AIF1 and IB4 staining. Blue arrows indicate merged activated microglial cells with vascular endothelial cells. (A). Hu-EVs, (B). nBPD-EVs, (C). BPD-EVs. Scale bars: 25 µm. (D). Quantification of the intensity of neovascularization. (E). Quantification of the number of microglial cells (n = 5 retinas/group, ** p < 0.01, *** p < 0.001, ns: no significance). AIF1: Green; IB4: Red.
Figure 4. BPD-EVs increase Müller cell number and microglial cell activation in the retinas of neonatal mice. (A–C). Representative images of neonatal mouse retinas stained for Müller cells (GRAP+, Green) following adoptive transfer of control Hu-EVs, nBPD-EVs, and BPD-EVs. (D–F). Representative images of retinal vasculature stained with IB4 (Red). (G–I). Representative images of activated microglial cells stained with CD206 (Blue). (J–L). Images of merged IB4, anti-GFAP and anti-CD206 staining. (A,D,G,J): control Hu-EVs control, (B,E,H,K): nBPD-EVs, (C,F,I,L): BPD-EVs. Scale bars: 70 µm. (M). Quantification of the intensity of GFAP staining. (N). Quantification of the number of activated microglial cells (CD206). (n = 3 retinas/group, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

3.3. Adoptively Transferred BPD-EVs Significantly Enhance the Expression of Transcripts Related to Inflammation and Angiogenesis in the Retinas of Neonatal Mice

To further evaluate the impact of BPD-EVs on neonatal retinas, we conducted an analysis of inflammation-related transcripts through qRT-PCR of neonatal mouse retinas. Our findings indicated that retinal transcript levels of genes encoding inflammation and pyroptosis/apoptosis-associated Casp3 and Casp8 were significantly upregulated by 1- to 2-fold in the mouse pups injected with BPD-EVs compared to those injected with nBPD-EVs or human EV controls (Figure 5A). Similarly, elevated transcript levels ranging from 3- to 4-fold were found for the inflammasome sensor Nlrp3 and adaptor Asc, as well as caspase substrate and pyroptosis effector Gsdmd (Figure 5B). Transcripts for the pro-inflammatory cytokines Il1b and Il6 were also elevated by approximately 10-fold and 2.5-fold (Figure 5C), while transcripts for Aif1 and Vegf were elevated nearly 2.5-fold (Figure 5D). These changes in mRNA transcripts are consistent with our immunofluorescent microscopy results showing elevated retinal inflammation and neovascularization after BPD-EV injection (Figure 2, Figure 3 and Figure 4). Since BPD-EVs were observed to upregulate the expression of retinal transcripts, including Casp3 and Casp8, which are associated with pyroptosis and apoptosis, we conducted TUNEL assays on human retinal microvascular endothelial cell (HRMEC) cultures exposed to plasma EVs. These assays revealed a statistically significant increase in cell death in the BPD-EV-treated cells compared to both nBPD-EV- and Hu-EV-treated cells (Figure 6C,F,G).
Figure 5. BPD-EVs significantly upregulate the transcripts of inflammation-related genes. (A). The transcripts of the pyroptotic/apoptotic effectors Casp3 and Casp8 were significantly upregulated by 2-fold compared to Hu-EVs. (B). The transcripts of the inflammasome-associated Nlrp3, Asc, and Gsdmd were significantly upregulated by 2-fold compared to Hu-EVs. (C). The transcripts of the inflammatory cytokines Il1β and Il6 were upregulated more than 10-fold and 2-fold compared to Hu-EVs. (D). The transcripts of Vegf and Aif1 were upregulated 2-fold compared to Hu-EVs (n = 5 retinas/group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: no significance).
Figure 6. Human retinal endothelial cell TUNEL assay. Human endothelial cell cultures were exposed to EVs for 48 h, fixed, and TUNEL assayed. (A,D). Hu-EVs, (B,E). nBPD-EVs, (C,F). BPD-EVs. Scale bar: 25 µm. (G). Quantification of the number of apoptotic cells for each group (n = 3 replicates, **** p < 0.0001). DAPI: Blue; TUNEL positive: Green.

3.4. Comparative Analysis of Label-Free Quantitative Proteomics of BPD-EVs and nBPD-EVs

To identify EV proteins and determine if any BPD-EV proteins are known mediators of inflammation and pathological angiogenesis, we conducted a comprehensive assessment of each EVs’ proteomic profile using LC-MS/MS proteomic analysis by comparing quantitative proteomic alterations between BPD-EVs and nBPD-EVs, as illustrated in Figure 7A. Our quantitative proteomics analysis identified a total of 560 unique proteins in EVs isolated from the plasma of both BPD and nBPD infants. Among these, 333 proteins were of high enough abundance to be quantified across our four BPD and four nBPD-EV samples. Gene Set Enrichment Analysis (GSEA) of these 333 proteins revealed quantitative differences between BPD and nBPD-EVs, with a majority of significantly elevated gene set biological pathways associated with BPD-EVs proteins. The normalized enrichment score (NES) was 3.22, indicating a markedly significant upregulation of these expressed protein networks in BPD-EVs and underscoring the distinct molecular profile of BPD-EV proteins in comparison to nBPD-EVs (Figure 7B).
Figure 7. Differential abundance analyses of EV proteomic data. (A). Protocol for EV proteomic analysis. (B). Gene Set Enrichment Analysis (GSEA) plot for coexpression, comparing BPD-EV vs. nBPD-EV proteomic profiles. Green line (enrichment curve): the running normal enrichment score (NES = 3.221, FDR q = 0.04951); bottom heat strip: the ranked protein list from most upregulated in BPD (left, red) to most upregulated in nBPD (right, blue). The blackest bars cluster on the left (red), indicating that the majority of coexpressed proteins are upregulated in BPD-EVs. (C). Volcano plot comparing BPD-EV and nBPD-EV proteins with the top up and down significantly differentially abundant proteins labeled by red and blue letters. The 333 proteins are ranked in the volcano plot according to their statistical p value (Y-axis) as log 10 and their relative abundance ratio (log2FC) between BPD-EVs (B) and control nBPD-EVs (A). The 25 red dots show the comparatively hyper-abundant proteins in BPD-EVs, the 31 blue dots represent the comparatively hypo-abundant proteins in BPD-EVs, and the gray dots are the equally abundant proteins. (D). Principal component analysis (PCA) was performed on 8 samples of the top fifty significantly changed proteins. The plot shows the separation of nBPD-EVs and BPD-EVs by PC1 and PC2. (E). A heat map showing the top 40 upregulated and downregulated BPD-EV and nBPD-EV proteins. The color in each tile represents the scaled abundance value (adjusted p < 0.05). (F). Western blots of vWF, TNC, IGFBP2, and CD81 for nBPD-EVs and BPD-EVs. (G). Quantification of western blots of vWF, TNC, and IGFBP2.
The differences in average EV protein abundances analyzed in a volcano plot revealed that 31 proteins were significantly downregulated and 25 were significantly upregulated in BPD-EVs compared to nBPD-EVs (Figure 7C and Table S2). Principal component analysis (PCA) of the top 50 significantly changed proteins in our eight EV samples demonstrated a distinct separation between BPD-EVs and nBPD-EVs, indicating significant alterations in the principal protein components of preterm infants’ BPD-EVs compared to nBPD-EVs (Figure 7D). A heatmap displaying the top 40 significantly upregulated or downregulated proteins from each of the four BPD- and nBPD-EVs is shown in Figure 7E and Table S3. The major significantly upregulated BPD-EV proteins include IGFBP2, TNC, myeloperoxidase (MPO), vWF, and CD5L, which are either implicated in inflammation and angiogenesis signaling pathways or in innate immunity (Table S4) [25,26]. Western blots additionally validated that IGFBP2, vWF, and TNC were elevated in BPD-EVs relative to nBPD-EVs using CD81 as an EV loading control (Figure 7F,G and Figure S2).

4. Discussion

BPD has been increasingly regarded as a multi-organ disease involving not only the lungs (BPD) but the brain (NDI) and eyes (ROP) [1,2,3]. Our previous studies have shown that plasma EVs from both hyperoxia-exposed rats and BPD infants can induce both lung and brain injury when a single dose of EVs is adoptively transferred into neonatal rats or mice [16,17]. In this study, we determined whether circulating BPD-EVs also contribute to retinal eye injury in neonatal mice with some characteristics of the ROP that occurs in BPD infants and investigated the potential molecular mechanisms involved. Like our previous studies, we found that the single dose of BPD-EVs we used can induce significant retinal injuries in the eyes of neonatal mice, although at levels less than those seen with the continuous exposure of neonatal mouse retinas to circulating EVs in the OIR mouse models or BPD infant retinas with ROP [16,17]. Our results show that circulating BPD-EVs, compared to nBPD-EVs, increase retinal microglia and Müller cell activation, promoting abnormal neovascularization and inflammation in neonatal mouse retinas. These findings were supported by qRT-PCR, which showed that transcripts of Vegf and Aif1 were upregulated in these mouse retinas but not in controls. Furthermore, there was a notable upregulation of mRNA transcripts for NLRP3 inflammasome-associated proteins, sensor Nlrp3, adaptor Asc, and pyroptosis effector Gsdmd, as well as pyroptosis/apoptosis-related genes (Casp3, Casp8).
When these results are considered with our previous studies in lung and brain [16,17] and our TUNEL assays here with cultured retinal vascular epithelial cells, they suggest that BPD-EVs may trigger retinal programmed cell death pathways such as apoptosis or pyroptosis in vivo. Whether apoptosis or pyroptosis is involved cannot be determined by the TUNEL assay, as DNA fragmentation is a feature of both cell death mechanisms. However, our previous reports showing that an anti-ASC antibody or knockout of GSDMD can alleviate retinopathy in OIR mice by reducing retinal inflammation and cell death does suggest that inflammasome-mediated pyroptosis may contribute the retinal injuries seen here for BPD-EVs.
When administered to neonatal mice, the BPD-EVs triggered a pronounced upregulation of genes critical to apoptosis, inflammasome activation, and inflammatory cytokine production within the retinas. This suggests that circulating BPD-EVs act as potent systemic mediators capable of transporting disease-associated signaling mediators that initiate retinal inflammation and abnormal twisted angiogenesis, characteristic of ROP phase II, in the developing retinas. The observed effects of BPD-EVs on abnormal proliferative neovascularization were less pronounced than those typically seen in OIR. This may be due to a lower amount of BPD-EVs administered to neonatal mice compared to the higher oxygen used in OIR. Also, it is unlikely that the effects of BPD-EVs are due to eliciting a cross-species immunological response in recipient mice, as human-adult EVs, which should be even more immunoreactive than neonatal EVs, had no ROP-inducing activity. Generally, EVs do not induce major immunological effects in vivo because they are rapidly cleared due to their small size, with reported half-lives of ~30 min to 12 h [23,27]. Additional studies have shown that human EVs can be administered to mice without inducing major immunogenicity, particularly in short-term or single-dose studies [28].
We performed proteomic profiling of BPD-EVs and nBPD-EVs by LC-MS/MS, with the proteomic analyses further substantiated for some proteins by western blot validation. Further co-expression data analysis using GSEA revealed that proteins present at higher levels in BPD-EVs are associated with biological functions or regulatory mechanisms relevant to BPD pathogenesis. Several of the hyper-abundant BPD-EV proteins are involved in inflammation, including DEFA1B, IGFBP2, CD5L, TNC, and vWF, suggesting that these BPD-EV proteins could contribute to the retinal inflammation and angiogenesis observed in our adoptive transfer experiment (Table S4). The abundances of IGFBP2, TNC and vWF were validated by western blots, but we did not conduct functional validation experiments such as depletion or supplementation to directly demonstrate that BPD-EV cargo proteins can initiate retinal inflammation or angiogenesis in this study. While additional functional studies are needed to validate the cargo protein roles, other researchers have reported that some of these proteins are involved in processes related to retinal inflammation, blood vessel formation, and neurodegeneration. For example, specifically, DEFA1B has been associated with innate immune-mediated orbital inflammation and lung inflammation involving microglia or neutrophils and epithelial cells with cytotoxic activity that induces cell death [29,30]. Similarly, elevated IGFBP2 levels have been reported in diabetic neovascular disease [20], were they are thought to promote pathological angiogenesis by upregulating proangiogenic factors such as VEGF-A via activation of signaling pathways [31]. Interestingly, researchers have also reported that IGFBP2-mediated NLPR3 inflammasome activation is involved in dry AMD pathogenesis [32]. CD5L has been reported to be expressed in retinal microglia and retinal epithelial cells in retinal diseases, where its presence modulates inflammatory cytokine responses and influences neutrophil-driven neovascularization [33]. TNC has been reported to be involved in glial cells and the inflammatory response in retinal angiogenesis, optic nerve degeneration and diabetic retinopathy [34,35]. vWF is released during endothelial cell damage, as both a marker and mediator of inflammation in vascular diseases including retinopathy [36,37,38]. The hyperabundance of vWF in BPD-EVs may be significant, as BPD is characterized by impaired vascular development due to vascular endothelial injury or activation, with accompanying inflammation, in both the BPD lungs and the ROP retinas [39,40,41]. Interestingly, three of these upregulated EV cargo proteins, DEFA1B, IGFBP2 and LCP1, have been reported to be non-traditional NLRP3 inflammasome activators [32,42,43], suggesting that they may mediate the BPD-EV-induced cell death observed in our retinal vascular epithelial cells (Figure 6). This is highly supportive of our previous studies suggesting BPD-EVs act via inflammasome-mediated pathways, as we found they contain ASC and GSDMD [16,17].
Our study has some limitations. EV sample sizes for both the BPD and nBPD groups were relatively small, although EV proteomic profile differences indicated a clear separation between the two groups. Future studies will enroll more patients in each group to confirm our findings and determine if there is a correlation between EV proteomic profiles and the severity of infant ROP. Also, the degree of neovascularization induced by the BPD-EVs, although significant, was less than usually seen with OIR. We will investigate whether increasing EV dosage or frequency or using shorter or longer endpoints leads to a greater degree of neovascularization, as well as our other measured parameters. Our findings are based on transcript upregulation and retinopathy analysis. In the future, we plan to perform more functional investigations, such as PERG and OCT, to test neonatal mouse retinal function. The presence of significantly elevated levels of unique proteins like DEFA1B, IGFBP2, TNC, vWF, LCP1, COL1A2, and CD5L in BPD-EVs suggests further functional investigation is needed to determine if these proteins could be potential biomarkers and/or therapeutic targets for BPD and ROP.
In summary, this study shows that EVs derived from BPD preterm infants exhibit a proteomic signature distinct from those from nBPD infants and suggests that BPD-EVs play a critical role in initiating inflammatory responses and promoting pathological angiogenesis in neonatal mouse retinas. It emphasizes the potential of BPD-EVs as mediators in ROP development. These findings are consistent with and expand on those studies linking plasma EVs from BPD infants with inflammation and organ damage in lungs and brains [17,44,45].

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cells15040367/s1, Figure S1: BPD-EVs increase microglial cell number in the retinas of neonatal mice; Figure S2: Full images of western blot for anti-CD81, TNC, vWF and IGFBP2; Table S1: Materials and Reagents; Table S2: Volcano plot for Figure 7C; Table S3: Heatmap for Figure 7E; Table S4: The role of significantly upregulated unique proteins in BPD-EVs.

Author Contributions

Conception and design of the study, H.Y. and S.W.; Acquisition, analysis, and interpretation of data, H.Y., S.C., M.R.D., M.B., A.S., K.Y., A.M.B., M.E.H. and S.W.; Drafting manuscript, H.Y., M.R.D. and S.W.; Editing manuscript, H.Y., M.R.D. and S.W. All authors have read and agreed to the published version of the manuscript.

Funding

The funding was provided by the NIH through grant number NIH R01HL156803 (S.W.), Batchelor Award (S.W.), Project Newborn (S.W.) and NIH R01EY105130 (M.E.H.).

Institutional Review Board Statement

This study was conducted in the neonatal ICU (NICU) at the Holtz Children’s Hospital in the Jackson Health System and the University of Miami Miller School of Medicine in accordance with the Declaration of Helsinki and approved by the Institutional Review Boards of Jackson Health System and the University of Miami Miller School of Medicine (protocol #20200736, 18 June 2020).

Data Availability Statement

Reviewers can access the dataset by logging in to the PRIDE website using the following account: Username: reviewer_pxd068467@ebi.ac.uk; Password: JnMKze1vbv6R.

Conflicts of Interest

All authors have no conflicts of interest to declare.

References

  1. Schmidt, B.; Asztalos, E.V.; Roberts, R.S.; Robertson, C.M.; Sauve, R.S.; Whitfield, M.F. Trial of Indomethacin Prophylaxis in Preterms (TIPP) Investigators. Impact of bronchopulmonary dysplasia, brain injury, and severe retinopathy on the outcome of extremely low-birth-weight infants at 18 months: Results from the trial of indomethacin prophylaxis in preterms. JAMA 2003, 289, 1124–1129. [Google Scholar] [CrossRef] [Scilit]
  2. Li, W.; Wang, Y.; Song, J.; Zhang, C.; Xu, Y.; Xu, F.; Wang, X.; Zhu, C. Association between bronchopulmonary dysplasia and death or neurodevelopmental impairment at 3 years in preterm infants without severe brain injury. Front. Neurol. 2023, 14, 1292372. [Google Scholar] [CrossRef] [Scilit]
  3. Bae, S.P.; Shin, S.H.; Yoon, Y.M.; Kim, E.K.; Kim, H.S. Association of Severe Retinopathy of Prematurity and Bronchopulmonary Dysplasia with Adverse Neurodevelopmental Outcomes in Preterm Infants without Severe Brain Injury. Brain Sci. 2021, 11, 699. [Google Scholar] [CrossRef] [Scilit]
  4. Kornacka, M.K.; Tupieka, A.; Czajka, I.; Gajewska, M.; Golebiewska, E. Tissue and blood oxygenation in newborns <1250 g and incidence of ROP. Ginekol. Pol. 2003, 74, 538–544. [Google Scholar]
  5. Smith, L.E. Pathogenesis of retinopathy of prematurity. Semin. Neonatol. 2003, 8, 469–473. [Google Scholar] [CrossRef] [Scilit]
  6. Fu, X.; Feng, S.; Qin, H.; Yan, L.; Zheng, C.; Yao, K. Microglia: The breakthrough to treat neovascularization and repair blood-retinal barrier in retinopathy. Front. Mol. Neurosci. 2023, 16, 1100254. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  7. Fevereiro-Martins, M.; Guimaraes, H.; Marques-Neves, C.; Bicho, M. Retinopathy of prematurity: Contribution of inflammatory and genetic factors. Mol. Cell Biochem. 2022, 477, 1739–1763. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  8. Fevereiro-Martins, M.; Marques-Neves, C.; Guimaraes, H.; Bicho, M. Retinopathy of prematurity: A review of pathophysiology and signaling pathways. Surv. Ophthalmol. 2023, 68, 175–210. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  9. Buzas, E.I. The roles of extracellular vesicles in the immune system. Nat. Rev. Immunol. 2023, 23, 236–250. [Google Scholar] [CrossRef] [Scilit]
  10. Buzas, E.I.; Gyorgy, B.; Nagy, G.; Falus, A.; Gay, S. Emerging role of extracellular vesicles in inflammatory diseases. Nat. Rev. Rheumatol. 2014, 10, 356–364. [Google Scholar] [CrossRef] [Scilit]
  11. Minciacchi, V.R.; Freeman, M.R.; Di Vizio, D. Extracellular vesicles in cancer: Exosomes, microvesicles and the emerging role of large oncosomes. Semin. Cell Dev. Biol. 2015, 40, 41–51. [Google Scholar] [CrossRef] [Scilit]
  12. Yang, Y.; Boza-Serrano, A.; Dunning, C.J.R.; Clausen, B.H.; Lambertsen, K.L.; Deierborg, T. Inflammation leads to distinct populations of extracellular vesicles from microglia. J. Neuroinflamm. 2018, 15, 168. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  13. Kumar, M.A.; Baba, S.K.; Sadida, H.Q.; Marzooqi, S.A.; Jerobin, J.; Altemani, F.H.; Algehainy, N.; Alanazi, M.A.; Abou-Samra, A.B.; Kumar, R.; et al. Extracellular vesicles as tools and targets in therapy for diseases. Signal Transduct. Target. Ther. 2024, 9, 27. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  14. Akers, J.C.; Gonda, D.; Kim, R.; Carter, B.S.; Chen, C.C. Biogenesis of extracellular vesicles (EV): Exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies. J. Neurooncol 2013, 113, 1–11. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  15. Cai, Y.; Chen, T.; Cai, Y.; Liu, J.; Yu, B.; Fan, Y.; Su, J.; Zeng, Y.; Xiao, X.; Ren, L.; et al. Surface protein profiling and subtyping of extracellular vesicles in body fluids reveals non-CSF biomarkers of Alzheimer’s disease. J. Extracell. Vesicles 2024, 13, e12432. [Google Scholar] [CrossRef] [Scilit]
  16. Starke, N.; Challa, N.V.D.; Yuan, H.; Chen, S.; Duncan, M.R.; Cabrera Ranaldi, E.; de Rivero Vaccari, J.P.; Schott, A.; Aguilar, A.C.; Lee, Y.S.; et al. Extracellular Vesicle ASC: A Novel Mediator for Lung-Brain Axis in Preterm Brain Injury. Am. J. Respir. Cell Mol. Biol. 2024, 71, 464–480. [Google Scholar] [CrossRef] [Scilit]
  17. Ali, A.; Zambrano, R.; Duncan, M.R.; Chen, S.; Luo, S.; Yuan, H.; Chen, P.; Benny, M.; Schmidt, A.; Young, K.; et al. Hyperoxia-activated circulating extracellular vesicles induce lung and brain injury in neonatal rats. Sci. Rep. 2021, 11, 8791. [Google Scholar] [CrossRef] [Scilit]
  18. Wu, S.; Benny, M.; Duara, J.; Williams, K.; Tan, A.; Schmidt, A.; Young, K.C. Extracellular vesicles: Pathogenic messengers and potential therapy for neonatal lung diseases. Front. Pediatr. 2023, 11, 1205882. [Google Scholar] [CrossRef] [Scilit]
  19. Yuan, H.; Chen, S.; Duncan, M.R.; de Rivero Vaccari, J.P.; Keane, R.W.; Dalton Dietrich, W.; Chou, T.H.; Benny, M.; Schmidt, A.F.; Young, K.; et al. IC100, a humanized therapeutic monoclonal anti-ASC antibody alleviates oxygen-induced retinopathy in mice. Angiogenesis 2024, 27, 423–440. [Google Scholar] [CrossRef] [Scilit]
  20. Welsh, J.A.; Goberdhan, D.C.I.; O’Driscoll, L.; Buzas, E.I.; Blenkiron, C.; Bussolati, B.; Cai, H.; Di Vizio, D.; Driedonks, T.A.P.; Erdbrugger, U.; et al. Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. J. Extracell. Vesicles 2024, 13, e12404. [Google Scholar] [CrossRef] [Scilit]
  21. Son, J.P.; Kim, E.H.; Shin, E.K.; Kim, D.H.; Sung, J.H.; Oh, M.J.; Cha, J.M.; Chopp, M.; Bang, O.Y. Mesenchymal Stem Cell-Extracellular Vesicle Therapy for Stroke: Scalable Production and Imaging Biomarker Studies. Stem Cells Transl. Med. 2023, 12, 459–473. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  22. Lai, C.P.; Tannous, B.A.; Breakefield, X.O. Noninvasive in vivo monitoring of extracellular vesicles. Methods Mol. Biol. 2014, 1098, 249–258. [Google Scholar] [CrossRef] [Scilit]
  23. Lai, C.P.; Mardini, O.; Ericsson, M.; Prabhakar, S.; Maguire, C.; Chen, J.W.; Tannous, B.A.; Breakefield, X.O. Dynamic biodistribution of extracellular vesicles in vivo using a multimodal imaging reporter. ACS Nano 2014, 8, 483–494. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  24. Fadl, B.R.; Brodie, S.A.; Malasky, M.; Boland, J.F.; Kelly, M.C.; Kelley, M.W.; Boger, E.; Fariss, R.; Swaroop, A.; Campello, L. An optimized protocol for retina single-cell RNA sequencing. Mol. Vis. 2020, 26, 705–717. [Google Scholar] [PubMed]
  25. Nakagawa, K.; Sakiyama, H.; Tsuchida, T.; Yamaguchi, K.; Toyoguchi, T.; Masuda, R.; Moriya, H. Complement C1s activation in degenerating articular cartilage of rheumatoid arthritis patients: Immunohistochemical studies with an active form specific antibody. Ann. Rheum. Dis. 1999, 58, 175–181. [Google Scholar] [CrossRef] [Scilit]
  26. Gragnano, F.; Sperlongano, S.; Golia, E.; Natale, F.; Bianchi, R.; Crisci, M.; Fimiani, F.; Pariggiano, I.; Diana, V.; Carbone, A.; et al. The Role of von Willebrand Factor in Vascular Inflammation: From Pathogenesis to Targeted Therapy. Mediat. Inflamm. 2017, 2017, 5620314. [Google Scholar] [CrossRef] [Scilit]
  27. Driedonks, T.; Jiang, L.; Carlson, B.; Han, Z.; Liu, G.; Queen, S.E.; Shirk, E.N.; Gololobova, O.; Liao, Z.; Nyberg, L.H.; et al. Pharmacokinetics and biodistribution of extracellular vesicles administered intravenously and intranasally to Macaca nemestrina. J. Extracell. Biol. 2022, 1, e59. [Google Scholar] [CrossRef] [Scilit]
  28. Zhu, X.; Badawi, M.; Pomeroy, S.; Sutaria, D.S.; Xie, Z.; Baek, A.; Jiang, J.; Elgamal, O.A.; Mo, X.; Perle, K.; et al. Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells. J. Extracell. Vesicles 2017, 6, 1324730. [Google Scholar] [CrossRef] [Scilit]
  29. Ganz, T. Defensins: Antimicrobial peptides of innate immunity. Nat. Rev. Immunol. 2003, 3, 710–720. [Google Scholar] [CrossRef] [Scilit]
  30. Ugurel, E.; Erdag, E.; Kucukali, C.I.; Olcay, A.; Sanli, E.; Akbayir, E.; Kurtuncu, M.; Gunduz, T.; Yilmaz, V.; Tuzun, E.; et al. Enhanced NLRP3 and DEFA1B Expression During the Active Stage of Parenchymal Neuro-Behcet’s Disease. In Vivo 2019, 33, 1493–1497. [Google Scholar] [CrossRef] [Scilit]
  31. Wang, F.; Zhang, L.; Sai, B.; Wang, L.; Zhang, X.; Zheng, L.; Tang, J.; Li, G.; Xiang, J. BMSC-derived leptin and IGFBP2 promote erlotinib resistance in lung adenocarcinoma cells through IGF-1R activation in hypoxic environment. Cancer Biol. Ther. 2020, 21, 61–71. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  32. Zhao, Y.; Zhang, Y.; Li, J.; Zhang, Y.; Qu, Y. The role of IGF2BP2 in macrophage-mediated NLRP3 inflammasome activation in the pathogenesis of dry AMD. Biol. Direct 2025, 20, 57. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  33. Iannaccone, A.; Hollingsworth, T.J.; Koirala, D.; New, D.D.; Lenchik, N.I.; Beranova-Giorgianni, S.; Gerling, I.C.; Radic, M.Z.; Giorgianni, F. Retinal pigment epithelium and microglia express the CD5 antigen-like protein, a novel autoantigen in age-related macular degeneration. Exp. Eye Res. 2017, 155, 64–74. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  34. Kubo, Y.; Ishikawa, K.; Mori, K.; Kobayashi, Y.; Nakama, T.; Arima, M.; Nakao, S.; Hisatomi, T.; Haruta, M.; Sonoda, K.H.; et al. Periostin and tenascin-C interaction promotes angiogenesis in ischemic proliferative retinopathy. Sci. Rep. 2020, 10, 9299. [Google Scholar] [CrossRef] [Scilit]
  35. Reinhard, J.; Roll, L.; Faissner, A. Tenascins in Retinal and Optic Nerve Neurodegeneration. Front. Integr. Neurosci. 2017, 11, 30. [Google Scholar] [CrossRef] [Scilit]
  36. Murray, P.I.; Young, D.W.; Aggarwal, R.K.; Blann, A.D. Von Willebrand factor, endothelial damage and ocular disease. Ocul. Immunol. Inflamm. 1993, 1, 315–322. [Google Scholar] [CrossRef] [Scilit]
  37. Hirai, H.; Yamashita, M.; Matsumoto, M.; Nishiyama, T.; Wada, D.; Okabe, N.; Mizusawa, Y.; Jimura, H.; Ueda, T.; Ogata, N. Alteration of plasma von Willebrand factor in the treatment of retinal vein occlusion with cystoid macular edema. PLoS ONE 2022, 17, e0264809. [Google Scholar] [CrossRef] [Scilit]
  38. Starke, R.D.; Ferraro, F.; Paschalaki, K.E.; Dryden, N.H.; McKinnon, T.A.; Sutton, R.E.; Payne, E.M.; Haskard, D.O.; Hughes, A.D.; Cutler, D.F.; et al. Endothelial von Willebrand factor regulates angiogenesis. Blood 2011, 117, 1071–1080. [Google Scholar] [CrossRef] [Scilit]
  39. Patrick, C.H.; Lazarchick, J.; Horn, B.S. Alterations in von Willebrand factor antigen in premature infants with respiratory distress syndrome and chronic lung disease. Ann. Clin. Lab. Sci. 1993, 23, 39–46. [Google Scholar]
  40. Luan, Y.; Ding, W.; Ju, Z.Y.; Zhang, Z.H.; Zhang, X.; Kong, F. Bone marrow-derived mesenchymal stem cells protect against lung injury in a mouse model of bronchopulmonary dysplasia. Mol. Med. Rep. 2015, 11, 1945–1950. [Google Scholar] [CrossRef] [Scilit]
  41. Huang, Z.; Lin, B.; Han, D.; Wang, X.; Zhong, J.; Wagenaar, G.T.M.; Yang, C.; Chen, X. Platelets are indispensable for alveolar development in neonatal mice. Front. Pediatr. 2022, 10, 943054. [Google Scholar] [CrossRef] [Scilit]
  42. Eichholz, K.; Tran, T.H.; Cheneau, C.; Tran, T.T.P.; Paris, O.; Pugniere, M.; Kremer, E.J. Adenovirus-alpha-Defensin Complexes Induce NLRP3-Associated Maturation of Human Phagocytes via Toll-Like Receptor 4 Engagement. J. Virol. 2022, 96, e0185021. [Google Scholar] [CrossRef] [Scilit]
  43. Joshi, H.; Almgren-Bell, A.; Anaya, E.P.; Todd, E.M.; Van Dyken, S.J.; Seth, A.; McIntire, K.M.; Singamaneni, S.; Sutterwala, F.; Morley, S.C. L-plastin enhances NLRP3 inflammasome assembly and bleomycin-induced lung fibrosis. Cell Rep. 2022, 38, 110507. [Google Scholar] [CrossRef] [Scilit]
  44. Young, K.; Benny, M.; Schmidt, A.; Wu, S. Unveiling the Emerging Role of Extracellular Vesicle-Inflammasomes in Hyperoxia-Induced Neonatal Lung and Brain Injury. Cells 2024, 13, 2094. [Google Scholar] [CrossRef] [Scilit]
  45. Ransom, M.A.; Blatt, A.M.; Pua, H.H.; Sucre, J.M.S. The emerging role of extracellular vesicles in bronchopulmonary dysplasia. Am. J. Physiol. Lung Cell Mol. Physiol. 2024, 326, L517–L523. [Google Scholar] [CrossRef] [Scilit]
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