A TGFB2/TNF-induced in vitro model of proliferative vitreoretinopathy (PVR) using ARPE-19 cells confirms nicotinamide as an inhibitor of EMT and VEGFA secretion

Yuqing Huang; Roland Meister; Migle Lindziute; Maximilian Binter; Jan Tode; Carsten Framme; Heiko Fuchs

doi:10.1371/journal.pone.0340614

. 2026 Jan 13;21(1):e0340614. doi: 10.1371/journal.pone.0340614

A TGFB2/TNF-induced in vitro model of proliferative vitreoretinopathy (PVR) using ARPE-19 cells confirms nicotinamide as an inhibitor of EMT and VEGFA secretion

Yuqing Huang ¹, Roland Meister ¹, Migle Lindziute ¹, Maximilian Binter ¹, Jan Tode ¹, Carsten Framme ¹, Heiko Fuchs ^1,^*

Editor: Andre van Wijnen²

PMCID: PMC12798965 PMID: 41529022

Abstract

Proliferative vitreoretinopathy (PVR) is a vision-threatening fibrotic retinal disorder characterized by the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells. In this study, we established a pathophysiologically relevant in vitro model by co-stimulating ARPE-19 cells with transforming growth factor beta 2 (TGFB2) and tumor necrosis factor-alpha (TNF), referred to as ‘TNT’, and evaluated the anti-fibrotic and anti-angiogenic effects of Nicotinamide (NAM), a vitamin B3 derivative previously reported to counteract fibrosis in various disease models. Confluent ARPE-19 cells were treated with TGFB2, TNF, or TNT for up to six days. EMT progression was assessed via immunocytochemistry, Western blotting, and collagen gel contraction assays. Live-cell imaging (LCI) combined with Hoechst 33342 nuclear staining and automated tracking using Fiji/TrackMate enabled real-time analysis of cell migration and multicellular aggregation. VEGFA secretion was quantified by ELISA. TNT stimulation induced synergistic EMT-like features, including cell elongation, directional migration, extracellular matrix (ECM) remodeling, gel contraction, and formation of multicellular aggregates. TrackMate-based analysis revealed coordinated nuclear migration under TNT conditions. VEGFA secretion was significantly elevated at early time points. NAM co-treatment reduced ECM protein expression (FN1, COL1A1), attenuated migration and contraction, and significantly lowered VEGFA release. This TNT-based ARPE-19 model represents a robust, live-cell-compatible in vitro system that mimics both fibrotic and pro-angiogenic aspects of PVR. It allows real-time assessment of EMT progression and is suitable for screening anti-fibrotic compounds. Our findings suggest that Nicotinamide mitigates both fibrotic and angiogenic responses in this model and may hold therapeutic potential for fibrotic retinal diseases.

Introduction

Proliferative vitreoretinopathy (PVR) is a severe, pathological fibrotic response that can develop as an aberrant consequence of retinal detachment repair surgery or ocular trauma. Although it occurs in approximately 5–10% of primary rhegmatogenous retinal detachment (RRD) cases, PVR remains the leading cause of surgical failure in retinal repair [1]. Clinically, it is characterized by the formation of fibrotic membranes on the epiretinal and subretinal surfaces, as well as within the vitreous cavity, leading to retinal traction, contraction, and ultimately recurrent detachment [2]. Despite extensive research, the exact mechanisms underlying PVR pathogenesis remain incompletely understood.

Currently, it is widely accepted that PVR involves complex processes including epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells, inflammatory responses, extracellular matrix (ECM) remodeling, and subsequent fibrotic membrane formation [3]. During EMT, RPE cells lose epithelial characteristics such as polarity and intercellular adhesion, undergo cytoskeletal reorganization, and gain migratory and contractile properties, accompanied by the secretion of ECM components like fibronectin and type I collagen. Collectively, these alterations contribute significantly to membrane formation and tractional retinal detachment [3,4].

Additionally, EMT in PVR is associated with elevated secretion of pro-angiogenic factors, notably vascular endothelial growth factor A (VEGFA), which plays a key role in pathological neovascularization. Indeed, increased VEGFA levels have been documented across various vitreoretinal diseases, and anti-VEGF therapies have become a standard treatment approach in clinical ophthalmology [5,6]. Nevertheless, the precise role of EMT-driven VEGFA secretion specifically within PVR pathogenesis remains poorly characterized and warrants further investigation.

In vitro models of PVR-associated EMT typically utilize immortalized or primary RPE cells, and EMT is commonly induced by exogenous cytokines such as transforming growth factor-β (TGFB2), tumor necrosis factor-α (TNF), interleukin-6 (IL6), fibroblast growth factor-2 (FGF2), Gremlin, and Factor Xa [7,8]. Among these, TGFB2, the predominant isoform in ocular tissues, is a well-established EMT inducer that activates both Smad-dependent and non-Smad pathways [9]. TNF, a key inflammatory mediator secreted by retinal microglial cells, can synergistically enhance TGFB2-induced signaling, thereby accelerating EMT, fibrosis, and pathological tissue remodeling [10–12]. While many EMT studies rely on single-cytokine stimulation, cytokine profiling of vitreous humor from PVR patients reveals a complex milieu of inflammatory and fibrotic mediators [13]. This suggests that single-factor models may not sufficiently replicate the multifactorial nature of EMT in vivo. To address this limitation, we established a more pathogenetic relevant in vitro model by co-treating confluent ARPE-19 cells with different concentrations of TGFB2 and TNF-α, hereafter referred to as ‘TNT’.

Nicotinamide (NAM), also known as niacinamide or vitamin B3, is a water-soluble derivative of vitamin B3 and a precursor of nicotinamide adenine dinucleotide (NAD⁺), a key cofactor in cellular redox reactions, DNA repair, and stress signaling. NAM has demonstrated anti-inflammatory, antioxidative, and anti-fibrotic properties in a range of disease models, including cancer, neurodegeneration, and tissue fibrosis [14,15]. Mechanistically, NAM is thought to act through the modulation of NAD ⁺ -dependent pathways and inhibition of SIRT1, thereby influencing gene expression and cellular stress responses involved in EMT progression [16]. Given its reported ability to inhibit TGFB2-induced epithelial-to-mesenchymal transition (EMT) and fibrosis in various cell types, we investigated whether NAM could similarly counteract the effects of combined TNF and TGFB2 (TNT) treatment in ARPE-19 cells.

In this study, we aimed to establish and characterize a TNT-based, high-throughput ARPE-19 live-cell imaging assay that recapitulates early retinal detachment–associated EMT and fibrotic remodeling. Using nicotinamide (NAM) as a proof-of-concept compound, we further show that reductions in TNT-driven nuclear migration allow early identification, within the first 10 h of live-cell imaging, of substances that prevent the formation of dense, multilayered contractile RPE aggregates, without the need for end-point analysis.

Materials and methods

Cell culture and cytokines treatments

The ARPE-19 human RPE cell line (ATTC #CRL-2302) was cultured in DMEM/F12 medium (Gibco #21331−020) supplemented with 1 x GlutaMAX™ (Gibco #3505−061), 5% FBS (Pan-Biotech #P30-3606), and 2% Penicillin-Streptomycin (Gibco #15140−122).

For cytokine treatments, 1 × 10⁵ ARPE-19 cells were seeded in 24-well plates with 0.5 mL of complete medium containing 5% FBS. After 24 hours, the medium was replaced with fresh medium containing 2% FBS, 28 nM Hoechst (Sigma # B2261-25MG), and one of the following conditions (unless stated otherwise): 10 ng/mL TGFB2 (ThermoFisher #100-35B-10UG), 5 ng/mL TNF (ThermoFisher #300-01-10UG), 20 mM Nicotinamide (Sigma-Aldrich #N0636-100G), or their combinations. Cells were cultured for up to six days, with medium replenished every 72 hours.

Live-cell imaging

Live-cell imaging (LCI) was performed using the BioTek® Lionheart™ FX automated microscope. Imaging settings were applied as previously described, with minor modifications [17]. After cytokine treatment, the 24-well plate was placed into the humidity chamber maintained at 37 °C and 5% CO₂. Imaging was conducted using the phase-contrast channel and a 4 × PL FL objective. Hoechst (28 nM) was directly added to the culture medium to achieve nuclear staining. Laser autofocus was performed in the phase-contrast channel to determine the focal plane, which was then used for imaging Hoechst-stained nuclei via the DAPI filter, to minimize prolonged blue light exposure [18]. To minimize further phototoxicity due to blue light exposure, the ‘LED intensity’ was set to level 6, with an ‘acquisition time’ of 70 ms, and the maximum gain of 24 was used. Default autofocus and autoexposure settings were applied in the phase-contrast channel, and images were acquired every 20 minutes over a 72–144-hour period.

Cell tracking

Cell migration was analyzed using the TrackMate plugin in Fiji (ImageJ) [19]. Time-lapse DAPI image sequences were first converted into 8-bit grayscale. The ‘Set scale’ tool was used to convert pixels into micrometers (µm), and, under ‘Image Properties’ the time interval was set to 20 minutes to reflect the acquisition rate.

For migration tracking, the Fiji Plugin ‘TrackMate’ was used. First, the nuclei in each image were detected, using the ‘StarDist’ detector plugin [20,21]. For tracking, the LAP tracker was applied, setting ‘Frame to Frame Linking’ to 20 μm, ‘Track segment splitting’ to 15 µm, and ‘Track segment merging’ to 15 µm. Finally, Cell migration trajectories were visualized with a temporal projection of 10 hours ‘backwards in time’ and color-coded according to instantaneous velocity (µm/min). The resulting videos were converted to MP4 video files.

ICC staining

For ICC staining, 1x10⁵ ARPE-19 cells were seeded on a 13 mm diameter slide coverslip (Glaswarenfabrik Karl Hecht #41001113) in each well of a 24-well plate with 0.5 mL complete medium. Cells were washed twice with PBS (Carl Roth #1058.1) before fixation with 4% paraformaldehyde (Carl Roth #P087.6) for 20 min at RT, followed by two additional 10 min washes in PBS. Blocking was conducted with a solution composed of 5% goat serum (Millipore #S26-100 ml), 0.02% Tween®20 (Sigma #P9416), and 0.01% Triton™X-100 (Sigma #X100) in PBS for 1 h at RT.

Cells were incubated overnight at 4°C with 1:1000 dilutions of Fibronectin 1 (FN1) rabbit mAb (Cell Signaling #26836S), COL1A1 rabbit mAb (Cell Signaling #72026S), or ZO-1 rabbit mAb (Cell Signaling #13663S) in blocking solution. After two washes with PBS for 10 min at RT, cells were incubated with AlexaFluor™488 goat anti-rabbit (Invitrogen #A11034, 1:2000) and Rhodamine-phalloidin (Invitrogen #R415, 4 µL/mL) in PBST (0.1% Tween®20 in PBS) for 2 h at RT. After three washes in PBS for 10 min at RT, the coverslips were mounted upside down on a slide with Roti®-Mount FluorCare DAPI (Carl Roth #HP20.1). Phase-contrast and fluorescence images were recorded with an Observer Z.1 microscope (Carl Zeiss) using the ZEN-Blue analysis software (Carl Zeiss). Representative images were chosen from three biological replicates.

Western blot

4 × 10⁵ ARPE-19 cells were cultured in 6-well plates with 2 mL of complete medium. After 24h, cells were treated with TGFB2, TNF, TNT, or control with or without NAM for five days before lysis. Cells were lysed in 1 × Laemmli Sample Buffer (Bio-Rad #1610737) supplemented with a 1 × protease inhibitor cocktail (Cell Signaling #5871S). The lysates were mixed with 2-mercaptoethanol (Sigma-Aldrich #60-24-2), denatured at 95°C for 5 min, and stored at −20°C. Protein samples were loaded onto TGX Stain-Free™ Fast-Cast™ polyacrylamide gels (Bio-Rad #1610181) and separated by electrophoresis at 80–120 V. Resolved proteins were transferred onto an ethanol-activated Mini-size LF PVDF membrane (Bio-Rad #10026934) using the Trans-Blot® Turbo™ Transfer System at 1.3 A, 25 V for 7 min, or 10 min for high-molecular-weight proteins. After transfer, total protein bands were visualized using the ‘Stain-Free blot’ option of the ChemiDoc™ Imaging System following 45 sec UV activation and automatic exposure acquisition. Membranes were blocked with 5% milk powder (Carl Roth #T145.2) in 1 × Tris-buffered saline (TBS) at room temperature (RT) for 1 h, followed by overnight incubation at 4°C with a 1:1000 dilution of Fibronectin 1 (FN1) rabbit mAb (Cell Signaling #26836S) and COL1A1 rabbit mAb (Cell Signaling #72026S). After two 5-minute washes in TBS, membranes were incubated with a 1:1000 dilution of goat anti-rabbit secondary antibody StarBright™ Blue 700 (Bio-Rad #12004162) at RT for one hour. Fluorescence signals were detected using the ChemiDoc™ MP Imaging System (Bio-Rad) at an excitation/emission wavelength of 660–720 nm. Image quantification and normalization were performed using ImageLab 6 software (Bio-Rad). Protein band intensities were normalized to the corresponding total protein signal instead of a housekeeping protein. The calculated densitometric ratio was performed from three to four biological replicates. Raw western blots detecting FN1 or Col1A1, and their corresponding total-protein blots used for normalization, are shown in S1, S2, S6 and S7 Figs.

ELISA assays

To assess VEGFA secretion, conditioned media from ARPE-19 cells were collected at 24, 48, and 72 hours and stored at −80 °C until analysis. Before measurement, samples were thawed and centrifuged at 14,000 × g for 5 minutes, and 3 × 100 µL aliquots of each sample were used for quantification. VEGFA levels were determined using the Human VEGF-165 Development Kit (TMB) (Peprotech #99-T10K) according to the manufacturer’s instructions. Absorbance was measured using a Tecan Spark® M10 plate reader. Each condition was analyzed in three technical replicates across three biological replicates (n = 3).

Gel contraction assay

Collagen gels were prepared according to the manufacturer’s instructions (Corning #354249), yielding a final collagen concentration of 2.5 mg/mL. A volume of 500 µL of the collagen mixture was added to each well of a 24-well plate and incubated at 37 °C for 30 minutes to allow gel polymerization. After gelation, 1 × 10⁵ ARPE-19 cells were seeded on top of each gel and cultured under standard conditions. After 24 hours, the medium was replaced with the cytokine-containing medium as described above. To initiate contraction, the gels were carefully detached from the well walls using a 200 µL pipette tip. The gel area was recorded daily over five days, and gel contraction was quantified indirectly by changes in the gel area using Fiji (ImageJ). For statistical analysis, three to five biological replicates were analyzed.

Data analysis and statistics

Statistical analysis was performed using ANOVA in GraphPad Prism (Version 9.2.0). Migration parameters were extracted via TrackMate and exported for further processing. Western blot band intensities were quantified using ImageLab 6 (Bio-Rad). A p-value of p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) was considered statistically significant.

Results

TGFB2 and TNF have a synergistic effect on EMT

In our previous studies, we characterized the effects of TGFB2 on ARPE-19 cells and primary human RPEs, demonstrating its role in promoting EMT-associated changes [17,22]. However, the vitreous humor contains a complex mixture of cytokines rather than isolated factors. Notably, cytokine profiling of vitreous humor samples has revealed the simultaneous presence of pro-fibrotic mediators such as TGFB2 and pro-inflammatory cytokines like TNF [23,24]. To better mimic this physiological context and explore potential synergistic effects, we investigated the combined influence of TGFB2 and TNF on EMT progression in ARPE-19 cells. Therefore, ARPE-19 cells were assigned to four experimental groups: control, TGFB2, TNF, and a combination of TGFB2 and TNF, hereafter referred to as ‘TNT’.

Phase-contrast imaging at 72 hours post-treatment revealed distinct morphological alterations. Compared to the other three groups, TNT-exposed cells exhibited pronounced elongation and a distinct directional alignment (Fig 1A). To further characterize these changes, immunocytochemistry (ICC) was performed to assess the expression and spatial distribution of the mesenchymal extracellular matrix proteins Fibronectin 1 (FN1) and Alpha-1 Type I Collagen (COL1A1). Phalloidin staining was used to visualize actin filament organization. COL1A1 expression in the TGFB2 and TNF groups closely resembled that of the control group, with no significant alterations in localization or intensity. In contrast, the TNT group displayed a strong COL1A1 signal with localized ECM aggregation. FN1 showed punctate deposition in the TNF group and more pronounced patchy aggregates in the TGFB2 group. In the TNT group, FN1 formed extensive network-like deposits, suggesting a more advanced stage of ECM remodelling. While F-actin fibers in the control group were radially organized around the nucleus, the TNF group exhibited a more disorganized structure. In the TGFB2 group, actin filaments appeared denser with some degree of parallel alignment. Remarkably, TNT-treated cells showed a filament network similar to the TGFB2 group but with more pronounced elongation and directional organization (Fig 1B).

Fig 1 — **(A)** The morphology of ARPE-19 cells after 3 days of treatment in control, TGFB2, TNF, and TGFB2 + TNF (TNT) exposed groups. The scale bar represents 500 µm. **(B)** Three days after treatment, cells were immunostained for COL1A1, FN1, and F-actin. The scale bar represents 50 µm. **(C)** Representative Western blot of FN1 and COL1A1 in ARPE-19 cells after five days of treatment. **(D)** The densitometric ratio of FN1 and COL1A1 protein was normalized to total proteins. The values were normalized to the control group. The raw western blot images used for quantification are shown in Supplementary Information (S 1 and S 2 Figs). One-way ANOVA with Tukey’s multiple comparisons test (n = 4) was performed. The error bars represent SD. Ns, non-significant, **p < 0.01, ***p < 0.001, ****p < 0.0001.

To quantify protein expression, Western blot analysis was performed for FN1 and COL1A1. FN1 levels were significantly elevated in both TGFB2- and TNF-treated cells, with slightly higher expression observed in the TGFB2 group (Fig 1C, D). The TNT group exhibited the highest FN1 expression, significantly surpassing all other groups. Similarly, COL1A1 protein levels were markedly increased in the TNT group. While slight elevations were also observed in the TGFB2 and TNF groups, these did not reach statistical significance when compared to the control.

TNT remarkably enhances the mobility of ARPE-19 cells and promotes the formation of multi-layered cell clusters

To further explore the impact of the treatments on cellular dynamics, we next conducted live-cell imaging combined with Hoechst staining to monitor migration patterns and multicellular behavior in real-time. Cells in the TGFB2 group exhibited elongation and partial alignment compared to the control, 72 hours after cytokine exposure (Fig 2A). In the TNF group, elongation was more pronounced, accompanied by the formation of multicellular aggregates. Notably, the TNT group displayed not only multicellular aggregates but also substantial cellular aggregation, occasionally extending beyond 100 µm, as revealed by nuclear staining.

Fig 2 — **(A)** Phase contrast images, fluorescence images of Hoechst 33342-stained nuclei, and merged images of ARPE-19 cells after three days of treatment with TGFB2, TNF, or TNT are shown. The scale bar represents 500 µm. **(B)** Representative nuclear trajectory and velocity maps of ARPE-19 cells under the indicated treatment conditions at 24, 48, and 72 hours. Trajectories were reconstructed over the 10 h preceding each time point using the TrackMate/StarDist pipeline, and track colour encodes instantaneous nuclear velocity (µm/min) according to the scale bar. Apparent black regions within areas of high track density (marked by asterisks) correspond to densely packed nuclei in multicellular aggregates, which could no longer be segmented individually by the detection algorithm. The correspondence between these regions and Hoechst-positive multicellular aggregates is illustrated in S 3 Fig, S 2 and S 3 Videos.

To track nuclear migration, we used the FIJI plugin TrackMate in combination with the StarDist detector, which enabled robust and automated tracking of Hoechst-stained nuclei in LCI sequences over 10-h time windows preceding each time point. Our analysis revealed that TNT-treated cells exhibited rapid migration along well-defined trajectories, a behavior not observed in the other three groups (S2 and S3 Videos). Interestingly, the convergence of these cells at defined aggregation sites reflects a pattern characteristic of coordinated group migration. These results confirm that the TNT group exhibited significantly enhanced migratory activity compared to the other conditions (Fig 2B). For visual clarity, nuclei were hidden in the main trajectory maps so that tracking paths remained unobstructed. In TNT-treated wells, nuclei frequently formed dense, multilayered aggregates, at which point TrackMate could no longer reliably segment individual nuclei, resulting in apparent ‘gaps’ in the track maps. To make this explicit, we include trajectories reconstructed over the 10 h preceding the 0 h, 24 h, 48 h, and 72 h time points together with the nuclei overlay from a representative TNT experiment (S3 Fig).

Next, we examined how varying TNF concentrations (0, 1, 5, and 10 ng/mL) at a constant TGFB2 concentration of 10 ng/mL influence the formation of multicellular aggregates (S4 Fig). The 4 × 4 phase-contrast image montages, acquired three days post-exposure using a 4 × objective for improved overview, reveal a dose-dependent modulation of aggregate formation, such that the severity of the phenotype can be tuned according to the experimental objective.

To determine whether these differences in aggregate formation are preceded by changes in cell motility, we performed a separate tracking experiment in which confluent ARPE-19 cells were exposed to different ranges of TNF concentrations (0, 1, 5, and 10 ng/mL) in the presence of 10 ng/mL TGFB2. Migration trajectories and velocities were retrospectively analyzed over the 10 h preceding the 24 h, 48 h, and 72 h time points (S5 Fig). The tracking data indicate that, within the analysed 10 h windows, nuclear trajectories are faster and more persistent at higher TNF concentrations, consistent with enhanced collective migration before any detectable detachment.

TNT significantly enhances the contraction ability of ARPE-19 cells

LCI over five days of TNT treatment revealed pronounced morphological changes indicative of an EMT phenotype. In some instances, cells detached from the well bottom following TNT exposure, as documented in the Supplementary Video (S1 Video). A Collagen I-based GCA was performed to quantitatively assess their associated contractile properties.

After 72 hours of treatment, the gel area in the TGFB2 group showed a slight reduction compared to the control, while the TNF group exhibited a more pronounced decrease. Notably, the TNT group displayed the strongest contraction, with all differences reaching statistical significance (Fig 3A, B). To assess contraction dynamics, gel areas were monitored over five days, revealing distinct temporal patterns among the groups.

Fig 3 — **(A)** The Images of collagen gel contraction under different treatments (control, TGFB2, TNF, and TNT) over three days. The scale bar represents 1 cm. **(B)** The normalized area ratio of collagen I gel contraction after three days of treatment. The values were normalized to the control group. One-way ANOVA with Tukey’s multiple comparisons test (n = 3) was performed. The error bars represent SD. ns, non-significant, **p < 0.01, ***p < 0.001, ****p < 0.0001 **(C)** This collagen gel contraction area (cm² ± SEM) over 5 days for different treatment groups.

In the control group, contraction progressed at a relatively constant rate. In contrast, all three treatment groups exhibited the most pronounced gel shrinkage between day one and day two, followed by only minor changes from day four onward (Fig 3C).

Nicotinamide suppresses the TNT-induced EMT process in ARPE-19 cells

Next, we analyzed the impact of NAM on cell morphology, EMT marker expression, and TNT-induced migration and formation of multicellular aggregates. Phase-contrast microscopy and Hoechst staining revealed that TNT-treated cells exhibited pronounced elongation, multilayered structures, and aggregation, indicative of EMT progression. In contrast, NAM co-treatment attenuated cell elongation and multilayering, resulting in a morphology comparable to the control group. Live-cell imaging further demonstrated that, while TNT-treated cells underwent significant contraction and detachment over five days, NAM co-treatment preserved adhesion and minimized contraction (Fig 4A).

Fig 4 — **(A)** The morphology of ARPE-19 cells and nuclear staining after three days of treatment with control, control + NAM, TNT, and TNT + NAM. The scale bar represents 500 µm. **(B)** Cells were immunostained for COL1A1 and FN1 after 3 days of treatment. The scale bar represents 50 µm. **(C)** Representative Western blot of FN1 and COL1A1 in ARPE-19 cells after 5 days of treatment. **(D)** The densitometric ratio of FN1 and COL1A1 protein was normalized to total proteins. The values were normalized to the control group. One-way ANOVA with Tukey’s multiple comparisons tests (n = 3) was performed. The error bars represent SD. ns, non-significant, *p < 0.05, ***p < 0.001, ****p < 0.0001. The raw Western blot images used for quantification are shown in Supplementary Information (S 6 and S 7 Figs).

ICC revealed a marked increase in FN and COL1A1 deposition in the TNT-treated group, characterized by dense, fibrous alignment within the ECM. Co-treatment with NAM significantly reduced the fluorescence intensity of both proteins and disrupted their alignment, indicating an inhibitory effect on ECM remodeling (Fig 4B). These findings were corroborated by WB analysis, which confirmed that FN1 and COL1A1 expression levels peaked under TNT treatment but were significantly reduced with NAM co-treatment (Fig 4C, D). Consistent with these ECM changes, ZO-1 immunostaining after 5 days showed that TGFB2 and, more prominently, TNF and TNT disrupted the continuous junctional ZO-1 belt and induced a more cytoplasmic staining pattern. NAM co-treatment preserved a near-cobblestone, membrane-associated ZO-1 distribution in control and TGFB2 groups and partially restored junctional ZO-1 under TNF. Under TNT conditions, junctional ZO-1 was largely lost and only faintly detectable, whereas discontinuous membrane staining reappeared in the TNT + NAM group (S8 Fig).

Our LCI tracking analysis further revealed that NAM reduced the migration speed of both control and TNT-treated cells over 72 hours, while cells exposed to TNT alone retained the rapid, directional migration culminating in aggregation, as previously described (Fig 5A, S 4 Video).

Fig 5 — **(A)** Representative cell migration velocity maps of ARPE-19 cells treated with control, control + NAM, TNT, and TNT + NAM for 24 hours. The color scale represents migration velocity, ranging from 0.0 to 1.0 μm/min. The regions (marked by white asterisks) represent cell aggregates. These black areas appear due to the failure of gating by the StarDist detection algorithm. **(B)** Representative images of collagen gel contraction under different treatments after three days. The scale bar represents 1 cm. **(C)** The normalized area ratio of collagen I gel contraction after three days of treatment. One-way ANOVA with Tukey’s multiple comparisons tests (n = 5) was performed. The error bars represent SD. ns, non-significant, ****p < 0.0001.

In line with these observations, GCA assays showed that TNT-treated cells exhibited the strongest contractile activity, whereas NAM-exposed TNT-treated cells showed reduced contraction dynamics resulting in a significantly reduced gel shrinkage (Fig 5B, C).

Nicotinamide suppresses TNT-induced VEGFA secretion in ARPE-19 cells

Since EMT is not only associated with fibrotic remodeling but also with enhanced secretion of pro-angiogenic factors such as VEGFA, and given that VEGFA is a key target of several translational strategies in ophthalmology, we sought to determine whether TNT treatment modulates VEGFA secretion in ARPE-19 cells. VEGFA secretion levels were quantified via ELISA assays in the culture medium collected at 24, 48, and 72 hours post-treatment (Fig 6).

Fig 6 — Released VEGFA protein (pg/mL) in different groups after 24, 48, and 72 h of treatment was quantified by ELISA. Two-way ANOVA with Tukey’s multiple comparisons test (n = 3) was performed. The error bars represent SD. ns, non-significant, *p < 0.05 **p < 0.01, ***p < 0.001, ****p < 0.0001.

Compared to the control group, VEGFA secretion was significantly increased at 24 h, 48 h, and 72 h in both the TGFB2 and TNT groups, whereas a significant elevation was observed in the TNF group only after 72 h. Additionally, we investigated the effect of NAM on TNT-induced VEGFA secretion in ARPE-19 cells. The results showed that VEGFA secretion at all three time points was significantly lower in the TNT + NAM group compared to the TNT group alone, with all differences reaching highly statistical significance.

Discussion

PVR is a complex, multifactorial fibrotic retinal disorder characterized by epiretinal/subretinal formation of fibrotic membranes, primarily driven by EMT of RPE cells. While EMT of RPE cells plays a central role, PVR progression involves multiple contributing factors, including inflammation, proliferation, migration of diverse cell populations, and alterations in extracellular matrix remodeling. During PVR, RPE cells lose epithelial polarity and adhesion, gain contractile and migratory capabilities, and actively participate in fibrotic membrane formation. These transformations are orchestrated by a complex interplay of cytokines and growth factors present within the ocular microenvironment, including interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), placental growth factor (PlGF), vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), and transforming growth factor-β2 (TGF-β2) [24–27].

However, many in vitro models rely on single-cytokine stimulation, which may insufficiently capture the multifactorial nature of PVR pathogenesis. Recent work has begun to address this gap: in adult primary human RPE (ahRPE), co-stimulation with TGFB1 and TNF synergistically induces EMT, aggregate formation, and membrane contractility via p38 signaling, recapitulating key PVR features in vitro [28]. Similarly, Boles et al. showed that TNT treatment enhances EMT primary human RPE cells as well as aggregate formation and identified core regulatory factors through epigenomic and transcriptomic analyses [29]. Nonetheless, while primary hRPE cells are physiologically relevant, they pose challenges in terms of accessibility and experimental reproducibility.

To address this limitation, we established a co-treatment model using TNT in ARPE-19 cells, a well-characterized, readily available human RPE cell line. Although ARPE-19 cells differ phenotypically from primary or iPSC-derived RPE, they offer advantages in terms of reproducibility, culture stability, and scalability for mechanistic and screening studies. However, as an immortalized and partially dedifferentiated cell line, ARPE-19 cells may exhibit an EMT-like baseline phenotype and altered cytokine responsiveness compared with native or iPSC-derived RPE, which could lead to an overestimation of the absolute magnitude of EMT/fibrotic responses and inhibitory effects observed in our model. While models based on primary or iPSC-derived RPE or more complex cytokine environments may better recapitulate the in vivo pathophysiology of PVR, they are often experimentally demanding and less suited for high-throughput approaches. The TNT-ARPE-19 system represents a practical and informative platform for dissecting EMT dynamics, particularly in early-stage exploratory research. Notably, consistent with prior reports in primary human RPE cells [28,29], we also observed robust multicellular aggregate formation under TNT co-treatment in ARPE-19 cells, indicating that this aggregation phenotype is reproducible and can be translated from primary hRPE to this widely used cell line.

For real-time monitoring, we used live-cell imaging and automated cell tracking, integrating Hoechst nuclear staining with TrackMate (Fiji) [18,19]. This approach was designed to elucidate how directed migration and local cell–cell interactions give rise to multicellular aggregate formation. This LCI approach further enabled high-resolution visualization of dynamic cellular changes, including morphological alterations, as well as contraction and rapture events often observed in the TNT-treated group (S1 Video). TrackMate analysis using Fiji (Formerly ImageJ) further revealed that TNT treatment significantly enhanced directed cell motility. Notably, a marked increase in migration speed was already detectable at 24 hours in the TNT group, and comparing the TNT trajectories with those under NAM + TNT at this early time point allowed us to infer impending aggregate formation (trajectory convergence and local densification of tracks in TNT). A directional migration pattern resembling collective cell migration was observed, characterized by coordinated movement and alignment of neighboring cells, ultimately leading to the formation of multicellular cell aggregates. In addition, collagen contraction assays revealed a significant increase in contractile activity following TNT treatment, reinforcing the functional transition associated with EMT in this model. Thus, within the first 24 hours, LCI allows to infer whether a test compound attenuates TNT-induced aggregate formation, without the need to await endpoint assays.

Importantly, we observed a concentration-dependent effect of TNF, with lower levels resulting in reduced motility and cluster formation (S3 and S4 Figs). This finding underscores the modulatory role of inflammatory cytokines in EMT intensity and suggests that TNF may act as a para-inflammatory amplifier of fibrosis in the ocular environment. Such dose-sensitive effects have implications for understanding PVR severity and for the development of stage-adapted therapeutic interventions.

In addition to mechanistic insights, our study explored the therapeutic potential of nicotinamide (NAM), a vitamin B3 derivative with known anti-fibrotic and anti-inflammatory effects [30–35]. While NAM has previously been shown to inhibit EMT in adult hRPE by modulating transcriptional regulators [29], its role in other RPE models, such as ARPE-19 cells, has not been well defined.

In ARPE-19 cells, we show that NAM significantly suppressed TNT-induced EMT features, including cell elongation, ECM deposition, migration, contractility, and importantly, VEGFA secretion. The latter is particularly relevant given the dual role of EMT in promoting both fibrosis and pathological angiogenesis in retinal diseases.

In summary, our study establishes the TNT-ARPE-19 system as a robust and pathophysiologically relevant in vitro model that recapitulates key fibrotic and pro-angiogenic features of proliferative vitreoretinopathy. Co-stimulation with TGFB2 and TNF synergistically induced EMT-like phenotypes, including cytoskeletal remodeling, extracellular matrix deposition, increased contractility, multicellular aggregation, and elevated VEGFA secretion. Notably, the model allows real-time tracking of cell migration and phenotypic transitions using live-cell imaging and automated nuclear tracking, enabling early identification of EMT dynamics without reliance on endpoint-based assays. The fibrotic phenotype can be modulated by adjusting TNF concentrations, allowing for the simulation of mild to aggressive disease trajectories.

NAM significantly attenuated both fibrotic and angiogenic responses, supporting its potential as a modulator of EMT in retinal pigment epithelial cells. While the model does not replicate the full complexity of the vitreous microenvironment, it captures essential aspects of PVR pathomechanisms and provides a reproducible and scalable platform for mechanistic studies and compound screening under defined conditions. The use of the immortalized ARPE-19 cell line ensures consistency across experiments and facilitates high-throughput approaches. A further limitation of the present study is that we did not perform a dedicated, systematic viability assay under all treatment conditions, although initial propidium iodide staining did not reveal overt TNT-induced cell death. Future refinements may include the addition of further cytokines such as IL6, IL8, or IFNG to better approximate inflammatory profiles associated with different PVR subtypes. Moreover, validation of the observed effects in primary RPE cells or more complex co-culture and 3D models could enhance the physiological relevance and inform translational applications.

Supporting information

S1 Fig. Uncropped Western blot membranes used for COL1A1 detection in Fig 1C.

(A) RAW Western blot image showing COL1A1 protein used for densitometric quantification presented in Fig 1C. Each lane represents protein lysates from ARPE-19 cells treated under the indicated conditions. (B) RAW Western blot image showing total protein bands used for densitometric quantification presented in Fig 1C. Each lane represents protein lysates from ARPE-19 cells treated under the indicated conditions.

(PDF)

pone.0340614.s001.pdf^{(264.8KB, pdf)}

S2 Fig. Uncropped Western blot membranes used for FN1 detection in Fig 1C.

(A) RAW Western blot image showing FN1 protein used for densitometric quantification presented in Fig 4C. Each lane represents protein lysates from ARPE-19 cells treated under the indicated conditions. (B) RAW Western blot image showing total protein bands used for densitometric quantification presented in Fig 4C. Each lane represents protein lysates from ARPE-19 cells treated under the indicated conditions.

(PDF)

pone.0340614.s002.pdf^{(270.2KB, pdf)}

S3 Fig. Migration velocity maps of ARPE-19 cells under TNT stimulation.

Color-coded migration velocity maps of ARPE-19 cells exposed to TNT for 24, 48, and 72 hours. Nuclear trajectories were tracked 10 h backwards in time using live-cell imaging and analyzed with the StarDist/TrackMate plugin in FIJI. Hoechst-stained nuclei are shown in grey. The scale bar represents 1000 µm.

(PDF)

pone.0340614.s003.pdf^{(862.1KB, pdf)}

S4 Fig. Phase-contrast images of ARPE-19 cells after cytokine treatment.

Representative phase-contrast images of ARPE-19 cells three days after treatment with 10 ng/mL TGFB2 and varying concentrations of TNT (0, 10, 5, and 1 ng/mL TNF in combination with 10 ng/mL TGFB2). The scale bar represents 2000 µm.

(PDF)

pone.0340614.s004.pdf^{(481.7KB, pdf)}

S5 Fig. Velocity-coded nuclear migration trajectories of ARPE-19 cells exposed to different TNF concentrations at a constant TGFB2 level.

Confluent ARPE-19 monolayers were treated with control medium (Con), 10 ng/mL TGFB2 + 10 ng/mL TNF, 10 ng/mL TGFB2 + 5 ng/mL TNF, 10 ng/mL TGFB2 + 1 ng/mL TNF, or 10 ng/mL TGFB2 alone and subjected to live-cell imaging. Hoechst-stained nuclei were tracked with StarDist/TrackMat plugin in FIJI, and trajectories were reconstructed for a 10-h tracking window ending at the indicated 24 h, 48 h, and 72 h time points. Tracks are color-coded for instantaneous migration velocity according to the heat map (0–1.0 µm/min). Scale bar: 1000 µm.

(PDF)

pone.0340614.s005.pdf^{(1MB, pdf)}

S6 Fig. Uncropped Western blot membranes used for COL1A1 detection in Fig 4C.

(A) RAW Western blot image showing COL1A1 protein used for densitometric quantification presented in Fig 4C. Each lane represents protein lysates from ARPE-19 cells treated under the indicated conditions. (B) RAW Western blot image showing total protein bands used for densitometric quantification presented in Fig 4C. Each lane represents protein lysates from ARPE-19 cells treated under the indicated conditions.

(PDF)

pone.0340614.s006.pdf^{(277.2KB, pdf)}

S7 Fig. Uncropped Western blot membranes used for FN1 detection in Fig 4C.

(PDF)

pone.0340614.s007.pdf^{(245.5KB, pdf)}

S8 Fig. ZO-1 immunocytochemistry after 5-day TGFB2/TNF ± NAM treatment.

ARPE-19 monolayers were exposed for 5 days to control medium (con), TGFB2, TNF, or the combination of TGFB2 + TNF (TNT) in the absence (left panel, “without NAM”) or presence (right panel, “with NAM”) of 20 mM nicotinamide (NAM). Cells were fixed and stained for ZO-1 (green) and counterstained with DAPI (blue); merged images are shown in the right column of each panel. TNT corresponds to 10 ng/mL TGFB2 + 5 ng/mL TNF. Images are representative of three independent experiments. Scale bar: 50 µm.

(PDF)

pone.0340614.s008.pdf^{(306.6KB, pdf)}

S1 Table. Raw data: All individual data points obtained from the respective experiments and used for the generation of Figs 1 D, 3 B, C, 4D, 5C, and 6A, B are provided.

(XLSX)

pone.0340614.s009.xlsx^{(20.3KB, xlsx)}

S1 Video. Phase-contrast live-cell imaging of control and TNT-treated ARPE-19 cells.

Phase-contrast live-cell imaging of control and TNT-treated ARPE-19 cells. After 72 h of pre-exposure to control medium or TNT (10 ng/mL TGFB2 + 5 ng/mL TNF), images were acquired every 20 minutes over an additional 72 h. TNT-treated cells display pronounced epithelial-mesenchymal transition (EMT)-related morphological changes and frequent cell detachment events, which are not observed in control cells. Scale bar: 1000 μm.

(MP4)

Download video file^{(63.7MB, mp4)}

S2 Video. Live-cell imaging of Hoechst-stained nuclei in control and TNT-treated ARPE-19 cells.

Following three days of pretreatment, nuclei were imaged every 20 minutes for 72 hours. Control cells show limited motility, whereas TNT-treated cells exhibit directional migration and aggregate formation. Scale bar: 1000 μm.

(MP4)

Download video file^{(51.7MB, mp4)}

S3 Video. Tracking of nuclear migration velocity in control and TNT-treated ARPE-19 cells.

Nuclear trajectories were detected using StarDist and tracked using the TrackMate plugin in Fiji. TNT-treated cells demonstrate significantly increased migration speed and directional movement toward aggregation points compared to controls. The color scale represents instantaneous velocity (0 to 1.0 μm/min). For clarity, nuclei are hidden in the visualization, and trajectories are shown retrospectively for the last 10 hours.

(MP4)

Download video file^{(35.8MB, mp4)}

S4 Video. Tracking of nuclear migration velocity in TNT-treated and TNT + NAM-treated ARPE-19 cells.

The upper row shows live-cell imaging (LCI) of Hoechst-stained nuclei used for tracking. The lower row displays the corresponding nuclear trajectories, detected using StarDist and analyzed via the TrackMate plugin in Fiji. TNT-treated cells exhibit significantly increased migration speed and directional movement toward aggregation points, while co-treatment with Nicotinamide (NAM) reduces both motility and aggregation. Scale bar: 1000 μm. The color scale represents instantaneous velocity (zero –1.0 μm/min).

(MP4)

Download video file^{(34.7MB, mp4)}

Data Availability

All relevant data are within the manuscript and its Supporting information files. Raw data for Figures are listed in Supplementary Table ST1 raw data.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0340614.r001

Decision Letter 0

Andre van Wijnen

31 Jul 2025

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Reviewer #1: Dear author�

In this manuscript, the authors investigated theTGFB2/TNF-induced in vitro model of Proliferative Vitreoretinopathy (PVR) using ARPE-19 cells. This study has generated some interesting data, however, this manuscript lacks some key data.

1.There is no internal control in the western blot shown in Figure 1 and 4.

2. Some data about mechanisms related to the induction of PVR by TGFB2/TNF are lacking in the text.

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Reviewer #1: Yes: Xiaodong chen

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PLoS One. 2026 Jan 13;21(1):e0340614. doi: 10.1371/journal.pone.0340614.r002

Author response to Decision Letter 1

29 Aug 2025

Dear Prof. Xiaodong Chen,

We would like to thank you for the constructive feedback on our manuscript “A TGFB2/TNF-Induced in vitro model of Proliferative Vitreoretinopathy (PVR) using ARPE-19 cells confirms Nicotinamide as an inhibitor of EMT and VEGFA secretion”.

We carefully addressed all comments raised during the review process. Below, we provide a point-by-point response highlighting the changes implemented in the revised version of the manuscript.

Reviewer’s comment 1:

There is no internal control in the western blot shown in Figure 1 and 4.

Response:

Thank you for pointing this out. Instead of using a conventional loading control such as β-actin or GAPDH, in this study we normalized all western blot data to total protein levels, which were detected by stain-free imaging. This technique directly visualizes the total protein in each lane based on the intrinsic fluorescence of tryptophan residues, providing a reliable and unbiased reference for normalization. Representative stain-free images are now included for reference.

Unfortunately, this was not clearly described in the submitted version of the manuscript. We have therefore revised the Methods section to explicitly state:

“After transfer, total protein bands were visualized using the ‘Stain-Free blot’ option of the ChemiDoc™ Imaging System following 45 s UV activation and automatic exposure acquisition.”

“Protein band intensities were normalized to the corresponding total protein signal instead of a housekeeping protein.”

For reasons of space and figure clarity, these stain-free blots are not shown in the main figures but the corresponding raw stain-free total protein blots have been included in the Supplementary Figures SF3–SF6 to ensure full transparency.

In addition, as requested by the Editor, all raw data underlying the presented figures have been provided as a separate supplementary file Supplementary Table ST1 -raw data.xlsx.

Reviewer’s comment 2:

Some data about mechanisms related to the induction of PVR by TGFB2/TNF are lacking in the text.

Response:

We appreciate this insightful comment. In the present study, our main objective was to establish a reliable model of PVR induction by TGFβ2/TNF, and we therefore focused primarily on characterizing the phenotypic and functional changes in this model. We did not perform detailed mechanistic experiments at this stage. However, we have revised the Discussion to better contextualize our findings within known signaling mechanisms. Specifically, we now emphasize that TGFβ2 predominantly signals via Smad2/3 phosphorylation to drive EMT and extracellular matrix production, whereas TNF activates NF-κB and MAPK pathways to enhance inflammation and amplify fibrotic responses. Their combined stimulation therefore creates a synergistic pro-fibrotic environment that models central mechanisms thought to contribute to PVR pathogenesis. We further highlight that nicotinamide attenuates both TGFβ2/Smad- and TNF/NF-κB–associated responses in our system, suggesting a dual inhibitory action across multiple signaling axes.

The revised text is included in the Discussion, stating:

“Mechanistically, TGFβ2 and TNF activate distinct but converging signaling cascades that jointly drive EMT and fibrosis in RPE cells. TGFβ2 primarily signals via Smad2/3 phosphorylation, promoting transcription of extracellular matrix components and EMT regulators, whereas TNF activates NF-κB and MAPK pathways, enhancing inflammatory gene expression and amplifying fibrotic responses. Their combined stimulation therefore provides a synergistic pro-fibrotic environment that reflects key aspects of cytokine-driven mechanisms implicated in PVR. Our data demonstrate that nicotinamide effectively attenuates both TGFβ2/Smad- and TNF/NF-κB–associated responses, suggesting that its inhibitory effect extends across multiple signaling axes. This dual blockade may underlie the robust suppression of EMT markers, contractility, and VEGFA secretion observed in our model.”

We believe that these revisions have significantly improved the clarity and completeness of our manuscript. We thank the reviewer for their valuable input and hope that the revised version meets the journal’s standards for publication.

Sincerely,

Heiko Fuchs

on behalf of all co-authors

Attachment

Submitted filename: Letter to the reviwer.docx

pone.0340614.s016.docx^{(115.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0340614.r003

Decision Letter 1

Andre van Wijnen

24 Sep 2025

Dear Dr. Fuchs,

Thank you for submitting your revised manuscript to PLOS ONE. The reviewers are supportive of your work, but still had some concerns that you should address to the best of your ability. Therefore, we invite you to submit a second revision of your manuscript that adequately addresses the points they raised. Requests for substantial additional experimentation that are not feasible can be addressed by acknowledging limitations in the current research design.

Please submit your revised manuscript by Nov 08 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Andre van Wijnen

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

Reviewer #1: The author mentioned “in methods section to explicitly state: “After transfer, total protein bands were visualized using the ‘Stain-Free blot’ option of the ChemiDoc™ Imaging System following 45 s UV activation and automatic exposure acquisition.” However�the corresponding raw stain-free total protein blots have not been included in the Supplementary Figures SF3–SF6. Please the author check and add or label raw stain-free total in the Supplementary Figures SF3–SF6.

Reviewer #2: comments uploaded as an attachment. Please see full comments from attachment

Comments to the authors

Authors have investigated PVR related EMT using ARPE-19 cell line and TGF-β and TNF-α induced EMT model. They have also tested nicotinamide potential to prevent EMT.

Abstract

-TNT is confusing and the text does not tell what it means. Please revise and make easy for reader to get meaning already from abstract.

Material and methods

- Need to fulfill reagent information e.g. supplier country information

- western blot line 177, complete cells were treated.

-add species of antibodies e.g. FN1.

- marking of the sample amount is confusing, lines 208-209 . Total sample 9? Please clarify into text.

- Add also concentration information to TGF-β and TNF-α.

- Why ANOVA was used even the sample amount was so small?

Results

- Make figures bigger and more visible.

- Supplementary video 1. Why TNT cell layer is damaged at the end? Is concentrations too high? In which timepoint other measurements have done? Are EMT markers formed due to death cells? Does video present migration? Basicly, when treat cells too high concentrations they always detach from cell plate well. Now figures and supplementary files are separated from text at the end, thus hard to follow exactly.

- Supplementary video SV2. Why are the nuclei accumulated almost on one side of the cell plate well? What it mean and what is the conclusion of it.

- Is sample amount three? Refering supplementary Table related to Fig 6. It is quite small sample amount. Is there done only on repetition with three samples? In figure legend, there should be mentione the sample amount (n).

-It would be interesting to see some comparing epithelial markers changes (e.g. occluding, E-cadherin, or ZO-1) to mesenchymal markers increase (e.g. α.SMA, N-cadherin, fibronectin, vimentin) after all treatments compared to control either in protein or gene expression levels.

- Fig 2, Why nuclei tend to be packed as densely. What does it mean? Make Fig 2 more visible. Now it is quite cloudy for interpret.

- Fig 2 B. Why is velocity scale (µm/min) with colours presented at the below of the figure? The figure does not really present any movement and the velocity at the below does not tell anything. Even colours presented in the velocity scale are not marked or presented in the figure. Please mark or explain velocity meaning or delete it under the figure and from legend. White dots represent aggregates. That should be shown also some concretic way. Now it is only showed by white dots which really not be as result. Formed aggregates should be proven to be formed at that site. It can just also be empty area.......

See rest comments from attachment

Reviewer #3: Summary

This manuscript presents a novel in vitro model of proliferative vitreoretinopathy (PVR) using ARPE-19 cells stimulated with TGF-β2 and TNF-α (termed "TNT"). This dual-cytokine approach successfully induces robust epithelial-mesenchymal transition (EMT), extracellular matrix remodeling, cell migration, collagen contraction, and VEGF-A secretion-key hallmarks of PVR pathophysiology. The authors demonstrate that nicotinamide (NAM) effectively attenuates these fibrotic and pro-angiogenic responses, supporting its therapeutic potential. The model is technically sound, amenable to live-cell imaging, and highly reproducible, offering a valuable platform for future compound screening efforts.

Minor points:

a.) Although the use of ARPE-19 cells is well-justified for establishing this screening model, the Discussion should more prominently acknowledge the inherent limitations compared to primary RPE cells or iPSC-derived RPE, particularly regarding translational relevance. For example, absence of melanin could affect cytokine responses and as as immortalized cells, ARPE-19 may already exhibit mesenchymal features that could overestimate EMT and therapeutic effects.

b.) While there are space limitations, some controls are very important to show in the main manuscript. Thus, including one representative stain-free Western blot in the main figures (alongside the supplementary data) would enhance transparency and allow readers to better assess loading controls and normalization approaches.

**********

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Reviewer #1: Yes: Xiaodong chen

Reviewer #2: No

Reviewer #3: No

**********

Attachment

Submitted filename: Comments to the authors.pdf

pone.0340614.s015.pdf^{(91.6KB, pdf)}

PLoS One. 2026 Jan 13;21(1):e0340614. doi: 10.1371/journal.pone.0340614.r004

Author response to Decision Letter 2

24 Nov 2025

Dear reviewers,

First, we thank the reviewers for taking the time to read and review our manuscript. We have improved the manuscript based on their comments and suggestions and would like to address their points in detail below.

Responses to Reviewer #1 comments

Reviewer #1 - additional comment (stain-free total protein blots):

“The author mentioned in methods section to explicitly state: “After transfer, total protein bands were visualized using the ‘Stain-Free blot’ option of the ChemiDoc™ Imaging System following 45 s UV activation and automatic exposure acquisition.” However, the corresponding raw stain-free total protein blots have not been included in the Supplementary Figures SF3-SF6. Please the author check and add or label raw stain-free total in the Supplementary Figures SF3-SF6.”

Response:

We thank the reviewer for carefully checking this point. In the original submission, the labelling and numbering of the supplementary figures were indeed not fully consistent with the description in the text, and the stain-free total protein images were not clearly identifiable.

In the revised version, we have corrected this and now provide, for each western blot experiment, both the raw immunoblot and the corresponding stain-free total protein blot as separate panels, with explicit labelling and highlighting the numbers of biological replicates. Specifically:

- Supplementary Figure SF1A-B: COL1A1 immunoblot and matching stain-free total protein blot for Figure 1C

- Supplementary Figure SF2A-B: FN1 immunoblot and matching stain-free total protein blot for Figure 1C

- Supplementary Figure SF6A-B: COL1A1 immunoblot and matching stain-free total protein blot for Figure 4C

- Supplementary Figure SF7A-B: FN1 immunoblot and matching stain-free total protein blot for Figure 4C

We have also updated the references to the supplementary material in the main text and Methods section so that the numbering now correctly points to these stain-free total protein controls. All corresponding densitometric data are included in Supplementary Table ST1 (Raw data).

We hope that this revised presentation fully addresses the reviewer’s concern regarding the availability and labelling of the stain-free total protein blots.

Responses to Reviewer #2 comments

Reviewer Comment 1 (Abstract):

“TNT is confusing and the text does not tell what it means. Please revise and make easy for reader to get meaning already from abstract.”

Response:

We thank the reviewer for this helpful remark. We agree that the abbreviation “TNT” should be introduced as clearly as possible in the abstract. We have therefore revised the relevant sentence to explicitly define the combined cytokine stimulus, which now reads as follows:

“Here, we establish a cytokine-driven in vitro PVR model by co-stimulating ARPE-19 cells with transforming growth factor beta 2 (TGFB2) and tumor necrosis factor-alpha (TNF); this combined TGFB2/TNF stimulus is hereafter referred to as ‘TNT’.”

We hope that this revised wording makes the meaning of the abbreviation immediately clear to the reader.

Reviewer Comment 2 (Materials and Methods – reagents):

“Need to fulfill reagent information e.g. supplier country information.”

Response:

We thank the reviewer for this comment and fully agree that transparent reagent information is essential for reproducibility. In the Materials and Methods section, we already provide the catalogue numbers (and, where applicable, the supplier names) for all reagents used. As these catalogue numbers are globally unique and independent of the supplier’s local branch or country, they allow readers worldwide to obtain the identical reagents without ambiguity. We therefore did not further specify supplier country information, as we consider the current level of detail sufficient to ensure reproducibility.

Reviewer Comment 3 (Western blot):

“Western blot line 177, complete cells were treated.”

Response:

We thank the reviewer for this helpful remark and agree that the original wording could be misinterpreted. We have therefore clarified the description in the Methods section to make explicit that intact ARPE-19 cells were treated prior to lysis. The revised sentence now states:

“After 24 h, cells were treated with TGFB2, TNF, TNT, or control with or without NAM for five days before lysis.” This wording should avoid any ambiguity regarding the treatment of complete cells before protein extraction.

Reviewer Comment 4 (Antibodies):

“Add species of antibodies e.g. FN1.”

We thank the reviewer for this helpful suggestion. We have revised the Methods section to explicitly state the host species of the primary antibodies. The respective sentence now reads:

“Membranes were blocked with 5% milk powder (Carl Roth #T145.2) in 1× Tris-buffered saline (TBS) at room temperature (RT) for 1 h, followed by overnight incubation at 4°C with a 1:1000 dilution of Fibronectin 1 (FN1) rabbit mAB (Cell Signaling #26836S) and COL1A1 rabbit mAB (Cell Signaling #72026S).”

Reviewer Comment 5 (Sample amount):

“Marking of the sample amount is confusing, lines 208–209. Total sample 9? Please clarify into text.”

Response:

We thank the reviewer for this comment. In the Materials and Methods section, we specify the replicate structure as follows:“Each condition was analyzed in three technical replicates across three biological replicates.”This indicates that three independent biological experiments were performed (n = 3), with each biological replicate measured in triplicate (technical replicates). To make the total number of biological replicates more immediately transparent for the reader, we now additionally state the number of biological replicates (n) in the respective figure legends and list them in detail in Supplementary Table ST1 (“Raw data”).

Reviewer Comment 6 (Concentrations) “Add also concentration information to TGF-β and TNF-α.”

Response:

We thank the reviewer for this helpful suggestion. We have already clarified the cytokine concentrations in the Materials and Methods section. The text states that ARPE-19 cells were treated with 10 ng/mL TGFB2 and 5 ng/mL TNF, unless otherwise indicated (e.g.SF 3+4).

Reviewer Comment 7 (Statistics):

“Why ANOVA was used even the sample amount was so small?”

Response:

We thank the reviewer for this comment. In our experiments we routinely compared 4–8 different conditions (control, TGFB2, TNF, TNT, with or without NAM). In such settings, a series of pairwise t-tests would markedly increase the risk of type I error, whereas one-way ANOVA is specifically designed to test for differences across multiple groups within a single model. Although the number of biological replicates per group is relatively small (n ≥ 3), this approach is widely used and appropriate for exploratory in vitro studies with multiple treatment conditions.

Reviewer Comment 8 (Results – Figures):

“Make figures bigger and more visible.”

Response:

We thank the reviewer for this helpful suggestion. In the revised manuscript, we have increased the sizes of Figure 2 and Figure 5 to improve visibility and interpretation. We would also like to note that the figures displayed in the review PDF generated by the PLOS ONE submission system are subject to automatic downscaling and compression, which can reduce their apparent resolution. The original figure files that we uploaded are high-resolution images, and in the final typeset version of the article, the publisher will use these full-resolution files so that readers can zoom in without a substantial loss of image quality.

Reviewer Comment 9 (Supplementary Video 1):

“Supplementary video 1. Why TNT cell layer is damaged at the end? Is concentrations too high? In which timepoint other measurements have done? Are EMT markers formed due to death cells? Does video present migration? Basicly, when treat cells too high concentrations they always detach from cell plate well.”

Response:

We thank the reviewer for these questions and for the careful inspection of Supplementary Video 1, and we apologize that an important piece of information was missing in the original legend. In the revised manuscript, we have now clarified that the video shows phase-contrast live-cell imaging of control- and TNT-treated ARPE-19 cells after 72 h of prior exposure to the respective conditions. From this time point onward, TNT-treated cells increasingly migrate and cluster into multicellular, multilayered aggregates with pronounced contractile behavior. The legend of Supplementary Video 1 has been updated accordingly.

The apparent “damage” of the TNT cell layer at later time points is therefore not due to acute cytotoxicity from excessively high cytokine concentrations, but reflects mechanically driven detachment of the monolayer caused by these contractile aggregates. As the aggregates pull on the surrounding sheet, the cell layer folds and partially lifts off the substrate, which is consistent with the membrane-like contraction described in proliferative vitreoretinopathy (PVR). In pilot experiments using propidium iodide (PI) staining, we did not observe an increased frequency of PI-positive nuclei under TNT conditions compared with controls, which is why we did not further pursue this readout in the final experimental series (data not shown). During the imaging period, we do not observe massive loss of cells or widespread nuclear fragmentation that would be expected with extensive cell death.

The cytokine concentrations used in our model (10 ng/mL TGFB2 and 5 ng/mL TNF, unless otherwise indicated) are within the range commonly applied in in vitro PVR/EMT models and were chosen because they robustly induce fibrotic remodeling without causing global cell loss. EMT and fibrotic markers (FN1, COL1A1, ZO-1 and F-actin reorganization) and VEGFA secretion were quantified at defined end points after TNT exposure (e.g. day 5), as specified in the Methods, i.e. at time points when TNT-treated cultures still consist of viable, contracting cell layers and aggregates. Thus, the increased EMT marker levels reflect active fibrotic remodeling rather than artefacts arising from dying cells.

Finally, Supplementary Video 1 indeed documents TNT-induced migration: cells undergo collective migration towards emerging foci, followed by three-dimensional piling-up into contractile aggregates. This behavior is further quantified and visualized in the main figures and supplementary figures, and the video is intended as a qualitative illustration of these TNT-induced migration and aggregation dynamics.

Reviewer Comment 10 (Supplementary Video 2):

“Why are the nuclei accumulated almost on one side of the cell plate well? What does it mean and what is the conclusion of it.”

Response:

We thank the reviewer for this observation. Supplementary Video 2 was recorded with a 4× objective in a fixed field of view (FOV) within a 24-well plate. At this magnification, the camera captures only a small fraction of the total growth surface, not the entire well. The apparent accumulation of nuclei “on one side of the well” therefore reflects local aggregate formation within this limited FOV rather than a global asymmetry of the whole well.

In replicate recordings, multicellular aggregates formed at multiple locations across the well. The lateral accumulation seen in the presented clip is thus a stochastic local event and illustrates the collective migration and clustering behavior induced under TNT conditions, which is consistent with the PVR-like phenotype we aim to model.

Reviewer Comment 11 (Sample size):

“Is sample amount three? Refering supplementary Table related to Fig 6. It is quite small sample amount. Is there done only on repetition with three samples? In figure legend, there should be mentione the sample amount (n).”

Response:

We thank the reviewer for this comment. For the experiments related to Figure 6 (and all other quantitative assays in this study), we performed three to five independent biological experiments. Thus, the data do not originate from a single experiment with three wells, but from three separate experimental runs. We now explicitly state the number of biological replicates (n) in the corresponding figure legends and in Supplementary Table ST1 (“Raw data”), so that the sample size is immediately transparent to the reader.

Reviewer Comment 12 (Epithelial vs. mesenchymal markers):

“It would be interesting to see some comparing epithelial markers changes (e.g. occluding, E-cadherin, or ZO-1) to mesenchymal markers increase (e.g. α.SMA, N-cadherin, fibronectin, vimentin) after all treatments compared to control either in protein or gene expression levels.”

Response:

We appreciate this thoughtful suggestion. Our a priori aim in the present study was to characterize fibrotic remodelling rather than to comprehensively stage EMT, which is why we focused on mesenchymal/fibrotic readouts such as FN1, COL1A1, and F-actin stress fibres that directly report matrix production and contractile reorganization relevant to PVR. We did not perform a broader panel of mesenchymal markers (e.g. α-SMA, N-cadherin, vimentin) or gene expression profiling in this work.

To address the reviewer’s comment, we additionally performed immunofluorescence staining for the epithelial tight-junction marker ZO-1 after five days of treatment. Under our culture conditions, robust junctional ZO-1 staining was only observed in confluent monolayers, which were consistently reached at day 5; therefore, this time point was chosen for epithelial marker analysis. The new data are presented in Supplementary Figure SF8. In control cells, ZO-1 shows a continuous junctional belt, whereas TNT treatment markedly disrupts this membrane-associated pattern. Co-treatment with nicotinamide partially preserves junctional ZO-1 staining compared with TNT alone, consistent with the overall EMT-attenuating and anti-fibrotic effect of nicotinamide in our model.

We did not analyse occludin or E-cadherin in this study, but agree that combining a broader epithelial and mesenchymal marker panel at the protein and/or transcript level would be an interesting objective for future work.

Reviewer Comment 13 (Fig. 2 – visibility & dense nuclei):

“Why nuclei tend to be packed as densely. What does it mean? Make Fig 2 more visible. Now it is quite cloudy for interpret.”

Response:

We thank the reviewer for this comment. As requested, we have enlarged Figure 2 in the revised manuscript to improve visibility. The densely packed appearance of the nuclei is an inherent feature of the TNT condition and reflects the underlying biology of our model: under TNT, ARPE-19 cells migrate and cluster into compact, multilayered aggregates rather than remaining in a simple monolayer. In a single-plane live-cell imaging readout, this three-dimensional piling-up of cells appears as closely apposed or overlapping nuclei within the field of view. Functionally, these multilayered, contractile aggregates are a key hallmark of our in vitro PVR system, as they are required to generate the pronounced tissue contraction and membrane-like behavior observed in Supplementary Video 1.

Reviewer Comment 14 (Fig. 2B – velocity scale & aggregates):

“Fig 2 B. Why is velocity scale (μm/min) with colours presented at the below of the figure? The figure does not really present any movement and the velocity at the below does not tell anything. Even colours presented in the velocity scale are not marked or presented in the figure. Please mark or explain velocity meaning or delete it under the figure and from legend. White dots represent aggregates. That should be shown also some concretic way. Now it is only showed by white dots which really not be as result. Formed aggregates should be proven to be formed at that site. It can just also be empty area in cell plate well for example due to death and detach cells. Should be proven to be aggregate. Otherwise need to be deleted from the figure and legend.”

Response:

We thank the reviewer for these helpful comments on Figure 2B. Regarding the velocity scale, Figure 2B displays nuclear migration tracks generated with the TrackMate/StarDist pipeline. The colour of each track segment encodes the instantaneous nuclear velocity (μm/min) according to the colour bar shown below the panel. In the original submission, this was not explained cle

Attachment

Submitted filename: Letter to the reviewersHF_211125.docx

pone.0340614.s017.docx^{(127.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0340614.r005

Decision Letter 2

Andre van Wijnen

23 Dec 2025

A TGFB2/TNF-Induced in vitro model of Proliferative Vitreoretinopathy (PVR) using ARPE-19 cells confirms Nicotinamide as an inhibitor of EMT and VEGFA secretion

PONE-D-25-33120R2

Dear Dr. Fuchs,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Academic Editor

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Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

**********

Reviewer #1: (No Response)

Reviewer #2: Authors have answered all my questions and improved manuscript based on my comments or justify if not. Manuscript is fine to be publish.

Reviewer #3: The authors have provided detailed responses to most of the reviewers’ comments, including improved figure labeling, expanded methodological clarity, and the addition of new data (e.g., ZO-1 staining) and supplementary imaging to support their interpretations. They have appropriately acknowledged the limitations of the ARPE-19 model, clarified the basis for their migration analyses versus cytotoxicity, and strengthened the Discussion to better position this system as a screening platform requiring validation in more physiologic models. Remaining issues are minor and largely editorial, and do not materially affect the scientific rigor or transparency of the work.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: Yes: Xiaodong chen

Reviewer #2: No

Reviewer #3: No

**********

PLoS One. doi: 10.1371/journal.pone.0340614.r006

Acceptance letter

Andre van Wijnen

PONE-D-25-33120R2

PLOS One

Dear Dr. Fuchs,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS One. Congratulations! Your manuscript is now being handed over to our production team.

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Associated Data

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

Supplementary Materials

S1 Fig. Uncropped Western blot membranes used for COL1A1 detection in Fig 1C.

(PDF)

pone.0340614.s001.pdf^{(264.8KB, pdf)}

S2 Fig. Uncropped Western blot membranes used for FN1 detection in Fig 1C.

(PDF)

pone.0340614.s002.pdf^{(270.2KB, pdf)}

S3 Fig. Migration velocity maps of ARPE-19 cells under TNT stimulation.

(PDF)

pone.0340614.s003.pdf^{(862.1KB, pdf)}

S4 Fig. Phase-contrast images of ARPE-19 cells after cytokine treatment.

(PDF)

pone.0340614.s004.pdf^{(481.7KB, pdf)}

S5 Fig. Velocity-coded nuclear migration trajectories of ARPE-19 cells exposed to different TNF concentrations at a constant TGFB2 level.

(PDF)

pone.0340614.s005.pdf^{(1MB, pdf)}

S6 Fig. Uncropped Western blot membranes used for COL1A1 detection in Fig 4C.

(PDF)

pone.0340614.s006.pdf^{(277.2KB, pdf)}

S7 Fig. Uncropped Western blot membranes used for FN1 detection in Fig 4C.

(PDF)

pone.0340614.s007.pdf^{(245.5KB, pdf)}

S8 Fig. ZO-1 immunocytochemistry after 5-day TGFB2/TNF ± NAM treatment.

(PDF)

pone.0340614.s008.pdf^{(306.6KB, pdf)}

S1 Table. Raw data: All individual data points obtained from the respective experiments and used for the generation of Figs 1 D, 3 B, C, 4D, 5C, and 6A, B are provided.

(XLSX)

pone.0340614.s009.xlsx^{(20.3KB, xlsx)}

S1 Video. Phase-contrast live-cell imaging of control and TNT-treated ARPE-19 cells.

(MP4)

Download video file^{(63.7MB, mp4)}

S2 Video. Live-cell imaging of Hoechst-stained nuclei in control and TNT-treated ARPE-19 cells.

(MP4)

Download video file^{(51.7MB, mp4)}

S3 Video. Tracking of nuclear migration velocity in control and TNT-treated ARPE-19 cells.

(MP4)

Download video file^{(35.8MB, mp4)}

S4 Video. Tracking of nuclear migration velocity in TNT-treated and TNT + NAM-treated ARPE-19 cells.

(MP4)

Download video file^{(34.7MB, mp4)}

Attachment

Submitted filename: Letter to the reviwer.docx

pone.0340614.s016.docx^{(115.7KB, docx)}

Attachment

Submitted filename: Comments to the authors.pdf

pone.0340614.s015.pdf^{(91.6KB, pdf)}

Attachment

Submitted filename: Letter to the reviewersHF_211125.docx

pone.0340614.s017.docx^{(127.7KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting information files. Raw data for Figures are listed in Supplementary Table ST1 raw data.

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PERMALINK

A TGFB2/TNF-induced in vitro model of proliferative vitreoretinopathy (PVR) using ARPE-19 cells confirms nicotinamide as an inhibitor of EMT and VEGFA secretion

Yuqing Huang

Roland Meister

Migle Lindziute

Maximilian Binter

Jan Tode

Carsten Framme

Heiko Fuchs

Roles

Abstract

Introduction

Materials and methods

Cell culture and cytokines treatments

Live-cell imaging

Cell tracking

ICC staining

Western blot

ELISA assays

Gel contraction assay

Data analysis and statistics

Results

TGFB2 and TNF have a synergistic effect on EMT

Fig 1. TGFB2 and TNF have a synergistic effect on Fibrosis.

TNT remarkably enhances the mobility of ARPE-19 cells and promotes the formation of multi-layered cell clusters

Fig 2. TNT enhances the mobility of ARPE-19 cells and promotes the formation of multi-layered cell clusters.

TNT significantly enhances the contraction ability of ARPE-19 cells

Fig 3. TNT significantly enhances the contraction ability of ARPE-19 cells.

Nicotinamide suppresses the TNT-induced EMT process in ARPE-19 cells

Fig 4. Nicotinamide suppresses TNT-induced EMT in ARPE-19 cells.

Fig 5. NAM inhibited TNT-induced migration and contractile activity in ARPE-19 cells.

Nicotinamide suppresses TNT-induced VEGFA secretion in ARPE-19 cells

Fig 6. Nicotinamide suppresses TNT-induced VEGFA secretion in ARPE-19 cells.

Discussion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Andre van Wijnen

Roles

Author response to Decision Letter 1

Decision Letter 1

Andre van Wijnen

Roles

Author response to Decision Letter 2

Decision Letter 2

Andre van Wijnen

Roles

Acceptance letter

Andre van Wijnen

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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