Experimental Eye Research 

Forkhead domain inhibitory-6 attenuates subconjunctival fibrosis in rabbit
model with trabeculectomy
The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian, China b Xiamen Eye Center, Xiamen University, Xiamen, China c Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu, Sichuan, China d Sichuan Provincial Key Laboratory for Human Disease Gene Study, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China,
Chengdu, China e Department of Ophthalmology, Shenzhen People’s Hospital, The 2ndClinical Medical College, Jinan University, Shenzhen, China f Institute of Laboratory Animal Sciences, Sichuan Academy of Medical Sciences & Provincial People’s Hospital, Chengdu, Sichuan, China g Shenzhen Key Laboratory of Ophthalmology, Shenzhen Eye Hospital, Jinan University, Shenzhen, China
ABSTRACT
Antiproliferative therapies are crucially important for improving the success rate of the glaucoma filtration
surgeries. In this study, we investigated the potential efficacy of Forkhead Domain Inhibitory-6 (FDI-6) in
inhibiting post-trabeculectomy subconjunctival fibrosis. In vitro, the effect of FDI-6 (10 μM) on fibrotic response
and its underlying mechanism were investigated in rabbit tenon’s fibroblasts (RTFs) treated with or without
transforming growth factor-β1 (TGF-β1, 20 ng/mL). In vivo, FDI-6 (40 μM) was injected subconjunctivally to a
rabbit trabeculectomy model. Intraocular pressure (IOP) changes were monitored within the 14-day period post￾surgery. Bleb morphology and subepithelial fibrosis at the operating area were evaluated with slit lamp and
confocal microscopic examinations and with histologic examinations. The results showed that, in cell culture
studies, FDI-6 suppressed the proliferation, migration, collagen gel contraction and the expression levels of
fibronectin (FN) and α-smooth muscle actin (α-SMA) in RTFs with TGF-β treatment by down-regulating the TGF-
β1/Smad2/3 signaling pathway. In animal studies, the IOPs of the FDI-6-treated group were significantly lower
than those of the saline-treated group after trabeculectomy. The FDI-6-treated eyes showed a better bleb
appearance with fewer blood vessels compared to the saline-treated eyes. The analysis of confocal microscopy in
vivo and histopathology revealed that subconjunctival fibrosis after trabeculectomy was significantly attenuated
in the FDI-6-treated group compared to the controls. In conclusion, our studies indicate that FDI-6 exerts an
inhibitory effect on subconjunctival fibrosis caused by trabeculectomy, holding potentials as a new anti￾proliferative agent used in anti-glaucoma filtration surgeries in the future.
1. Introduction
Glaucoma, characterized by progressive dysfunction and death of
retinal ganglion cells and their axons with elevated intraocular pressure
(IOP) as the major risk factor, is the leading cause of irreversible
blindness worldwide (Kang and Tanna Angelo, 2021; Davis et al., 2016;
Quigley and Broman, 2006). IOP lowering strategies including
medication, laser treatment and glaucoma filtration surgeries (e.g. tra￾beculectomy), have been proven to be effective interventions for glau￾coma (Paul, 2000; Lusthaus and Goldberg, 2019). Gene therapy is an
emerging approach with the potential to achieve long-term IOP-lower￾ing effect in the treatment of glaucoma (Tan et al., 2019, 2020).
Currently, trabeculectomy is still one of the most common procedures
used in the treatment of glaucoma (Gedde et al., 2018). This surgery
Corresponding author.
Corresponding author. Xiamen Eye Center, Xiamen University, Xiamen, China.
Corresponding author.
E-mail addresses: [email protected] (Y. Zhu), [email protected] (X. Liu), [email protected] (N. Fan). 1 The first four authors contributed equally to this paper.
Contents lists available at ScienceDirect
Experimental Eye Research
journal homepage: www.elsevier.com/locate/yexer

https://doi.org/10.1016/j.exer.2021.108725

Received 8 April 2021; Received in revised form 27 July 2021; Accepted 6 August 2021
Experimental Eye Research 210 (2021) 108725
2 creates a new fistula from anterior chamber to a subconjunctiva and
episcleral bleb, and therefore facilitates aqueous humor outflow and
lowers IOP. However, the long-term outcomes of this operation are not
always satisfactory mainly due to subconjunctival fibrosis of the surgical
site that may cause the obstruction of the artificial outflow channel and
failure of the surgery (Schlunck et al., 2016; Zada et al., 2018).
Subconjunctival fibrosis is a complex and multistage pathological
process with its underlying mechanism remained not fully clarified. At
the initial stage of the process, conjunctival fibroblasts differentiate into
myofibroblasts after the surgery. Then, myofibroblasts migrate, prolif￾erate, and synthesize the extracellular matrix proteins (ECM), such as
collagen type I alpha 1 chain (COL1A1) and fibronectin (FN), that
eventually result in excessive scarring and tissue fibrosis at the surgical
site. The fistula will be blocked, ultimately leading to failure of the
surgery (Klingberg et al., 2013; NikhalaShree et al., 2019; Shu and
Lovicu, 2017). Currently, antiproliferative agents including 5-fluoro￾uracil (5-FU) and mitomycin C (MMC) have been widely used clini￾cally during and after the surgery to prevent the formation of
subconjunctival fibrosis after the filtering surgeries (Bell et al., 2020;
Cabourne et al., 2015; Lama and Fechtner, 2003). Unfortunately, the
nonspecific effects of these agents may cause significant side effects such
as toxicity, hypotony, leakage or bleb-related infections in the
conjunctive, corneal and intraocular tissues (Higginbotham et al., 1996;
Mearza and Aslanides, 2007). Therefore, more efficient and safer com￾pounds are needed to specifically inhibit subconjunctival fibrosis.
Forkhead box M1 (FOXM1), a member of the forkhead/winged-helix
family of transcription factors, is mainly responsible for normal cell
proliferation, survival, and self-renewal as well as cancer initiation and
progression (Halasi and Gartel, 2013; Koo et al., 2012; Wang et al.,
2018). Its small-molecular inhibitor, Forkhead Domain Inhibitory-6
(FDI-6), can selectively bind to the DNA binding domain (DBD) of
FOXM1 and broadly repress the transcription of FOXM1-activated genes
which do not respond to genes regulated by other homologous forkhead
family factors (Gormally et al., 2014). Previous studies on FDI-6 mainly
focused on its anti-tumor activities in multiple cancers including breast
cancer (Gormally et al., 2014), liver cancer (Li et al., 2020) and lung
cancer (Kalinichenko and Kalin, 2015). The inhibition of FOXM1 is the
main mechanism underlying the antiproliferative activities of FDI-6.
Recently, there are accumulating evidences supporting the potential
function of FOXM1 on fibrotic diseases such as pulmonary fibrosis,
cardiac fibrosis and liver fibrosis. In those diseases, expression of
FOXM1 significantly increased, and its down-stream and effectors
signaling through multiple fibrosis related pathways, such as
TGF-β1/Smad, PI3K/Akt, p38 MAPK or canonical WNT/β-catenin, were
significantly up-regulated (Goda et al., 2020; Jeng et al., 2020; Katoh,
2018; Yang et al., 2016). However, there are no previous reports on the
application of FDI-6, a small-molecular inhibitor of FOXM1, in inter￾vention of fibrotic disorders.
In current study, the antifibrotic effect of FDI-6 and the possible
underlying mechanism were evaluated, especially FDI-6 on the TGF-β1-
induced fibrosis in RTFs in vitro and the trabeculectomy-induced sub￾conjunctival fibrosis in vivo. Our studies suggest that FDI-6 holds po￾tentials as a novel antiproliferative medication for prevention of scar
formation after trabeculectomy.
2. Materials and methods
2.1. Cell culture and treatments
Rabbit Tenon’s Fibroblasts (RTFs, iCell Bioscience Inc, China) were
cultured at 37 ◦C in a 5% CO2 incubator in primary fibroblast basal
medium containing 10% fetal bovine serum (FBS), 1% penicillin/
streptomycin and 1% primary fibroblast culture additive, and the me￾dium was further changed to primary fibroblast basal medium con￾taining 1% FBS to conduct the experiments. Cell medium was changed
daily. All experiments were performed on cells at passages 3 to 6. Cells
were divided into five groups and treated as follows: (1) blank control
group: cells were treated with medium only; (2) dimethyl sulfoxide
(DMSO, a solvent of FDI-6, diluted to 0.04% with culture medium)
group; (3) TGF-β1 (PeproTech, NJ, USA) group: cells were pretreated by
0.04% DMSO for 1 h followed by 20 ng/mL TGF-β1 treatment for
another 24 h (Chen et al., 2019; Han et al., 2019); (4) FDI-6+TGF-β1
group: cells were pretreated by 10 μM FDI-6 for 1 h followed by 20
ng/mL TGF-β1 for another 24 h; (5) FDI-6 (Sigma, MO, USA) group: cells
were treated 10 μM FDI-6 for 24 h; FDI-6 was dissolved in DMSO and
then diluted in culture medium to different concentration for following
experiments in RTFs. The morphology images were taken under a Leica
DM4 microscope (DM400B; Leica, Wetzlar, Germany). Each experiment
was repeated at least twice.
2.2. Cell counting kit-8 (CCK-8) assay
Cell Counting Kit-8 assay kit (Dojindo Molecular Technologies,
Kumamoto, Japan) was used to detect the proliferative and cell meta￾bolic ability of RTFs according to the manufacturer’s instructions.
Briefly, RTFs were seeded in 96-well plates at 4.5 × 103 cells per well
and treated with 2.5, 5, 10, 20, and 40 μM FDI-6, with or without 20 ng/
ml TGF-β1, respectively, for 24 h. The original culture medium was
discarded, and the fresh culture medium containing CCK-8 reagent (10
μl) was added to each well. After incubation for 3 h, the absorbance
value of each well at a wavelength of 450 nm (OD 450) was detected
using flame atomic absorption spectrophotometry (PerkinElmer, Bos￾ton, MA, USA).
2.3. Wound healing assay
RTFs in 12-well plates were cultured in growth medium at 5% CO2 at
37 ◦C for 24 h. When RTFs reached 80–90% confluence, the cell cultures
were scratched with the tip of the sterilized 200 μl pipette. Furthermore,
each well was washed three times with Dulbecco’s phosphate-buffered
saline (DPBS; Gibco-BRL, Grand Island, NY, USA) to remove the
debris, and the indicated drugs in serum-free medium were added to
each well. The images of cellular migration were taken at 0 h, 12 h and
24 h, respectively, and analyzed by ImageJ software (National Institutes
of Health, Maryland, USA). The percentage of wound healing in each
group was evaluated as follows: (area of original wound – area of actual
wound)/area of original wound × 100% (Santos et al., 2015). The cells
will grow into the scratched area at different rates upon the treatments.
When the cells at both sides of scratch edge contact each other and heal
the gap, the healing (wound closure) is reached.
2.4. Assay of collagen gel contraction
As described previously (Liu et al., 2008), Neutral type I collagen
from rat tail (A10483-01, Gibco, Thermo Fisher Scientific, former
savant, MA, USA) was prepared according to the manufacturer’s in￾structions, and was mixed with RTFs suspension (2.5 × 105 cells/ml in
medium) on ice in the volume ratio of 9:1. After coating with 1% bovine
serum albumin (BSA; Sigma, MO, USA) for 1 h at 37 ◦C, each well was
filled with 0.5 ml mixture in a 24-well culture plate, and the plate was
incubated for another hour to solidification. Collagen gels were isolated
from the sides of the wells with a sterilized 10-μl pipette tip. Serum-free
medium (0.5 ml) containing TGF-β1, DMSO, or FDI-6 was added on the
surface of each corresponding gel. Represent images of collagen gel
contraction were photographed by a Fluor ChemE (92-14860-00; Pro￾teinSimple, San Jose, CA, USA). The gel size was measured by Image J
software. The percentage of contraction area of each group was calcu￾lated as follows: (initial gel area – gel area at each time point)/initial gel
area × 100%.
Experimental Eye Research 210 (2021) 108725
2.5. Immunofluorescence
Cells were seeded to chambered slides and treated as described in
2.1. After 24 h post treatment, cells were fixed with 4% para￾formaldehyde (PFA) for 10 min, washed twice with PBS, and incubated
for 2 h in PBS containing 3% BSA and 0.3% TritonX-100 at room tem￾perature (RT). Next, cells were conjugated with primary antibody
against α-SMA (ab7817, mouse monoclonal antibody, 1:500; Abcam,
Cambridge, MA, USA), FN (ab6328, mouse monoclonal antibody, 1–5
μg/ml; Abcam, Cambridge, MA, USA) over night at 4 ◦C. Cells were
washed twice with PBS and incubated with an Alexa Fluor 594 sec￾ondary antibody (A201207, 1:1000; Invitrogen, Carlsbad, CA, USA) for
1 h at RT. For actin labeling, cells were directly incubated with
Rhodamine-phalloidin (PHDR1, 1:150; Cytoskeleton, Denver, CO, USA)
for 1 h at RT. After washed twice with PBS, cells were counterstained
with DAPI for 3 min and visualized by fluorescence microscopy
(DM400B; Leica, Wetzlar, Germany). Mean immunofluorescence in￾tensity (MFI) was analyzed using ImageJ software. Five 400-fold
magnified fields were randomly selected to count the MFI, and the
mean value was taken as the MFI value of each group.
2.6. Western blot
RTFs from all the groups were harvested after treatments while the
rabbits were sacrificed and harvested at day 14 post-operation (DPO 14),
respectively. For tissue harvesting, an 8 mm × 8 mm filtering area was
labeled by ponceau fuchsin. Then, the marked tissues containing con￾junctiva, sclera and choroid were separated and harvested intactly. Total
proteins were extracted using radio immunoprecipitation assay (RIPA)
lysis buffer (CWBio, Beijing, China) containing 1% protease inhibitor
cocktail (Sigma-Aldrich) and 1% phosphatase inhibitor (Sigma-Aldrich).
Protein samples (20–40 μg) were fractioned in a commercial poly￾acrylamide gel (4%–20% Mini-PROTEAN or Criterion TGX Precast Gel;
Bio-Rad, Hercules, CA, USA), and transferred to polyvinylidene fluoride
membrane (Millipore, Burlington, MA, USA). Membranes were blocked
in 5% nonfat dried milk in Tris-buffered saline with 0.1% Tween-20
(TBS-T) at RT for 1 h, and were incubated with the indicated primary
antibody against FOXM1 (ab207298, rabbit monoclonal antibody,
1:1000; Abcam, Cambridge, MA, USA), α-SMA (1:200), COL1A1
(ab6308, mouse monoclonal antibody, 1–2 μg/ml; Abcam, Cambridge,
MA, USA), FN (1–5 μg/ml), Smad3 (#9523, Rabbit monoclonal anti￾body, 1:1000, Cell Signaling Technology, Danvers, MA, USA), Phospho￾Smad2/smad3 (#8828, Rabbit monoclonal antibody, 1:1000, Cell
Signaling Technology, Danvers, MA, USA) or GAPDH (AF5718, goat
polyclonal antibody, 1 mg/mL; R&D Systems, Minneapolis, MN, USA)
overnight at 4 ◦C. After washing with 1 × TBS-T for 30 min, membranes
were incubated with corresponding secondary antibodies at room tem￾perature for 1 h. Bands were visualized with a Fluor ChemE (Pro￾teinSimple), and analyzed by ImageJ software (NIH, Bethesda, MD,
USA).
2.7. RNA isolation and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from cultured RTFs with Trizol Reagent
(Invitrogen, CA, USA). The absorbance values of RNA at OD 260 and OD
280 were detected using a spectrophotometer (Biophotometer plus,
Eppendorf, Germany) to calculate the RNA content, and reverse tran￾scription reaction was conducted using a kit (RevertAid First Strand
cDNA Synthesis Kit; Fermentas, Thermo Fisher Scientific, Pittsburgh,
PA, USA). SYBR Green Real-Time PCR Master Mix (Toyobo, Osaka,
Japan) was used for quantitative real-time PCR on a Rotor-Gene Q cycler
(QIAGEN, Germantown, MD, USA). The relative expression of target
genes was calculated using the 2− ΔΔCt method. Four primer pairs
designed and used for PCR amplification were as follows:
α-SMA forward, 5′
2.8. Animals
Adult New Zealand white rabbits (5 males and 5 females, 10–12
weeks old, 1.5–2 kg, the Institute of Laboratory Animal Sciences,
Sichuan Academy of Medical Sciences, Chengdu, China) were used in
this study. Animal studies were conducted in accordance with the As￾sociation for Research in Vision and Ophthalmology (ARVO) statement
for the Use of Animals in Ophthalmic and Vision Research, and approved
by the Institutional Animal Care and Use Committee of the Institute of
Laboratory Animal Sciences, Sichuan Academy of Medical Sciences and
Sichuan Provincial People’s Hospital.
The rabbits were anesthetized by intravenous injection with 3%
pentobarbital sodium (30 mg/kg, Sanofi, Paris, France). Trabeculec￾tomy was performed at the superior region of the right eye of the rabbit
by an experienced glaucoma specialist. Briefly, partial thickness 8-0 silk
suture was passed through the superior peripheral cornea and the eye
was infraducted. A limbus-based conjunctival flap was created in the
nasal-superior quadrant, and the sclera was exposed. Then, a square 3 ×
3 mm half thickness scleral flap was made. An internal bloc excision of
0.5 × 1 mm of tissue (trabeculectomy) followed by a peripheral iri￾dectomy were performed. The scleral flap was sutured with 10-0 nylon
sutures. The conjunctiva was closed with one single running 10-0 nylon
suture. Subconjunctival injection with 100 μl saline containing 0.17%
DMSO or 40 μM FDI-6 was conducted in surgical or non-surgical eyes in
supratemporal direction. The choice of 40 μM dose was selected based
on the result of a preliminary study, and this dose was able to decrease
the conjunctival scarring without adverse side effects. The animals were
topically applied with 0.3% ofloxacin ophthalmic ointment (Santen,
Inc., Tokyo, Japan) once a day and 0.3% ofloxacin eye drops (Minsheng
Pharma, Hangzhou, China) four times daily for 1 week. All surgeries
were performed by the same experienced ophthalmologist.
Rabbits were randomly grouped as follows (n = 4 eyes per group):
(1) blank control group (no medication, no surgery); (2) saline only
group (subconjunctival injection of 100 μl saline containing 0.17%
DMSO on the left eye); (3) surgery + saline group (subconjunctival in￾jection of 100 μl saline containing 0.17% DMSO on the right eye after
trabeculectomy, with the contralateral eye served as saline only group);
(4) FDI-6 only group (subconjunctival injection of 100 μl 40 μM FDI-6 on
the left eye); (5) surgery + FDI-6 group (subconjunctival injection of
100 μl 40 μM FDI-6 on the right eye after trabeculectomy; with the
contralateral eye served as FDI-6 only group). FDI-6 was dissolved in
DMSO and then diluted in saline to a 40 μM concentration (DMSO =
0.17%). Rabbits were treated with 100 μl saline containing 0.17% DMSO
or 40 μM FDI-6 by subconjunctival injection at DPO 0, DPO 1, DPO 3 and
DPO 5, and were followed up by IOP measurement and slit lamp
examination.
2.9. IOP measurement, bleb evaluation and corneal fluorescence staining
IOP was measured every other day during the 14-days period using
the TonoVet® rebound tonometer (Icare, Finland, Espoo, Finland). IOP
measurements were performed at the same time of the day between 2
and 4 p.m. by an experienced investigator. After anesthesia (3%
pentobarbital sodium, 30 mg/kg injected intravenously, Sanofi, Paris,
France), rabbits were examined clinically as described below. Bleb
morphology and the anterior segments were observed every day and
pictures were taken at DPO 7 and DPO 14 by slit lamp biomicroscope
(S350; Shanghai Medi Works Precision Instruments, Hangzhou, China).
In vivo confocal microscopy (HRT II/RCM, Heidelberg Engineering
GmbH, Heidelberg, Germany) was used to examine microcysts and the
presence of loose subepithelial connective tissue at the filtering area at
C. Lan et al.
Experimental Eye Research 210 (2021) 108725
4 DPO 14. These examinations were performed following the manufac￾turer’s recommended procedures, and no side effects related to these
examinations were found. At DPO 7 and DPO 14, bleb scoring was
evaluated with slit lamp examinations, according to Krofeld involving a
qualitative scale of 1+ to 4+, reflecting increasing bleb height and size
as follows: 1+, minimal height, conjunctiva thickening, and no micro￾cysts; 2+, microcysts are present; 3+, elevated bleb covering 3 to 4 clock
hours of the eye; and 4+, greatly elevated bleb covering longer than 5
clock hours of the eye (Perkins et al., 2002). A score of 0 indicates no
observable bleb. Bleb scoring was evaluated by an experienced
ophthalmologist. A fluorescein sodium ophthalmic strip was used for
corneal fluorescence staining, and the corneas of each rabbit were then
examined under a blue light of slit lamp biomicroscope at DPO 14.
2.10. Hematoxylin and eosin (H&E) and Masson’s trichrome staining
At DPO 14, rabbits were sacrificed using anesthesia overdose, both
eyes of which were then immediately enucleated. The areas of each bleb
were marked with pinkish red-soaked cotton (Wuhan Servicebio,
Wuhan, China), and the eyes were fixed in a formaldehyde, acetic acid,
and saline (FAS) fixative (Wuhan Servicebio, Wuhan, China) for 48 h,
embedded in paraffin, cut into sequential 4-μm tissue sections, and then
dewaxed with xylene. The sections were analyzed by H&E staining for
the observation of cellularity and Masson’s trichrome staining for
evaluation of collagen expression. Collagen expression of conjunctival
and scleral layer was quantified by collagen volume fraction (CVF) and
calculated as the ratio (%) of the area of blue-stained tissue to the total
area. Represent images were taken with a Leica DM4 microscope
(DM400B; Leica, Wetzlar, Germany).
2.11. Immunohistochemistry analysis
Tissue sections of rabbit eyes were made as described above and
incubated in goat serum for 30 min at RT. And then, the slices were
conjugated with primary antibody against α-SMA (1:500) overnight at
4 ◦C, followed by incubation with secondary antibodies conjugated to
peroxidase for 60 min at RT. Subsequently, the sections were stained
with 3,3′
-diaminobenzidine (DAB) and hematoxylin for microscopic
analysis (DM400B; Leica, Wetzlar, Germany).
2.12. Immunofluorescence analysis
Tissue sections of rabbit eyes were made as described above and
incubated in goat serum for 30 min at RT. And then, the slices were
conjugated with primary antibody against and COL1A1 (1:500) over￾night at 4 ◦C, followed by incubation with PE-labeled secondary anti￾bodies for 60 min at RT. Subsequently, the nuclei were counterstained
with DAPI for 3 min and visualized by a fluorescence microscopy
(DM400B; Leica, Wetzlar, Germany).
2.13. TUNEL assay
Tissue sections of rabbit eyes were made as described above and
processed with a TUNEL kit (In Situ Cell Death Detection Kit,
11684817910; Roche, Switzerland) according to the guidance of the
producer, and the images were analyzed using a fluorescence
microscopy.
2.14. Statistical analysis
All statistics were analyzed by SPSS 22 software (IBM-SPSS, Chicago,
IL, USA). Paired Student’s t-test was used to calculate statistical differ￾ences of matched two groups. When the variance was homogeneous,
one-way analysis of variance (ANOVA) followed by Tukey post hoc test
was used for comparison among three or more groups; Otherwise,
Tamhane’s T2 post hoc test was used. The p value of <0.05 was
considered significant. Data were presented as mean ± SEM for animal
studies and nonanimal studies using GraphPad Prism 6.
3. Results
3.1. FDI-6 inhibited FOXM1 expression and cell proliferation in cultured
RTFs
To examine the effects of FDI-6 on FOXM1 expression, the protein
level of FOXM1 in RTFs treated with various concentrations of FDI-6 was
detected. Our results showed that there was no significant difference of
FOXM1 expression among the blank control, DMSO, 2.5 and 5 μM FDI-6
groups (p > 0.05; n = 3 per group). However, compared to the DMSO
group, FOXM1 expression was significantly down-regulated in a dose￾dependent manner by 10–40 μM FDI-6 treatment for 24 h (Fig. 1A; p
< 0.05 in 10 μM, p < 0.01 in 20 and 30 μM and p < 0.001 in 40 μM versus
DMSO group; n = 3 per group). Moreover, to exclude the effects of drug
toxicity from FDI-6 on FOXM1 expression and optimize FDI-6 concen￾tration, total cell metabolic viability or cell proliferation was determined
by CCK-8 assay and cell counting. As showed in Fig. 1B, FDI-6 inhibited
cell metabolic viability/proliferation of RTFs, and the extent of inhibi￾tion increased with FDI-6 concentrations from 5 to 40 μM (p < 0.01 in 5
μM and p < 0.001 from 10 to 40 μM versus blank control; n = 3 per
group). The numbers of RTFs kept increasing after 5 μM FDI-6 treatment
for 24 h (p < 0.01 versus baseline, p < 0.05 versus blank control; n = 3),
remained a slight but non-significant increase at 10 μM (p > 0.05 versus
baseline, p < 0.01 versus blank control; n = 3), and decreased at higher
concentrations (20 and 40 μM; p < 0.01 in 20 μM and p < 0.001 in 40 μM
versus baseline, p < 0.001 in 20 and 40 μM versus blank control; n = 3 per
group), which indicated that cell death occurred (Fig. 1C). Together,
these data revealed that FDI-6 effectively inhibited FOXM1 expression,
and 10 μM appeared to be the optimal concentration for subsequent
studies in cultured RTFs.
3.2. FDI-6 reduced the TGF-β1-induced migration and proliferation of
cultured RTFs
FOXM1 silencing inhibited the TGF-β1-induced cellular proliferation
and migration (Zhang et al., 2019). To further investigate the anti￾fibrotic effects of FDI-6 in RTFs, the TGF-β1-induced (20 ng/ml) fibrosis
in RTFs was determined. The cellular migration ability was detected by
wound healing assay (Fig. 2A and B). There was no significant difference
of migration area (%) between the blank control group and the DMSO
group (p > 0.05; n = 3 per group). Compared to the DMSO group, the
migration area was decreased (22.8%; p < 0.001 at 24 h; n = 3) in the
10 μM FDI-6 group. Furthermore, RTFs showed an increased migration
area in the TGF-β1 group (11.5%; p < 0.05 at 24 h; n = 3), while FDI-6
reversed this increase dramatically (27.5%; p < 0.001 at 24 h; n = 3).
Additionally, CCK-8 assays were conducted to detect the cellular pro￾liferation ability at 24 h post-treatment (Fig. 2C). No significant differ￾ence of proliferation ability was found among the blank control, DMSO
and TGF-β1 groups (p > 0.05; n = 6 per group). However, the prolif￾eration ability significantly decreased by 69% (p < 0.001 versus TGF-β1
group; n = 6) and 62.7% (p < 0.001 versus DMSO group; n = 6) in the 10
μM FDI-6 group with or without TGF-β1 respectively. These results
indicated that FDI-6 was a potent suppressor of RTFs migration and
proliferation function.
3.3. FDI-6 decreased the TGF-β1-induced the collagen gel contraction of
cultured RTFs
The ability of fibroblasts to contract collagen gels was an important
marker of their differentiation into the myofibroblast phenotype and
was commonly related to fibrotic tissues (Nacu et al., 2008; Ngu et al.,
2014). In this study, the effects of FDI-6 on RTFs-mediated collagen gel
contraction were detected using a three-dimensional (3D) collagen gel
C. Lan et al.
Experimental Eye Research 210 (2021) 108725
5 system (Fig. 2D) as previously described (Liu et al., 2008). The mean
contraction area (%) of RTFs had no significant difference between the
blank control group and the DMSO group (p > 0.05; n = 3). However,
contraction was significantly reduced by 11.6% at 24 h in the FDI-6
group (p < 0.01 versus DMSO group; n = 3), suggesting a role of FDI-6
in modulation of myofibroblast activity. Moreover, the mean contrac￾tion area was increased by 7.7% over 24 h (p < 0.05 versus DMSO group;
n = 3) of stimulation in the TGF-β1 group, while this effect was reversed
by 15.2% (p < 0.001 at 24 h; versus TGF-β1 group; n = 3) with FDI-6
treatment. In addition, 12 h after FDI-6 treatment, the morphology of
RTFs was changed from a large and multinodular spindle to a small and
long spindle shape, consistent with the results at 24 h (Fig. 2E), indi￾cating an attenuation of cell-mediated local ECM remodeling.
3.4. FDI-6 prevented the TGF-β1-induced the expression of profibrotic
genes via the TGF-β1/Smad2/3 signaling pathway in cultured RTFs
The effects of FDI-6 on expression of the profibrotic genes including
α-SMA, COL1A1 and FN were tested by qRT-PCR and Western blotting.
The expression level of these genes in the DMSO group remained not
significantly different from the blank control group (p > 0.05; n = 4 per
group). Compared to the DMSO group, FDI-6 decreased expression of
these genes at both mRNA (Fig. 4B, p < 0.01 in COL1A1 and FN or p <
0.001 in α-SMA; n = 4 per group) and protein levels (Fig. 4C, p < 0.01 in
α-SMA, p < 0.001 in COL1A1 and FN; n = 3 per group) in cultured RTFs.
In contrast, there was an increased expression of these genes on both
mRNA (p < 0.05 in FN or p < 0.001 in α-SMA versus DMSO group; n = 4)
and protein levels (p < 0.01 in α-SMA or p < 0.001 in FN versus DMSO
group; n = 3) in the TGF-β1 group, where presence of FDI-6 (p < 0.01 or
p < 0.001 versus TGF-β1 group; n = 4 in mRNA analysis, n = 3 in protein
analysis) was reduced. Additionally, FOXM1 exhibited a similar
expression trend to these profibrotic proteins (Fig. 4A, p < 0.05 in FDI-6
group or p < 0.01 in TGF-β1 group versus DMSO group, p < 0.01 in FDI-
6+TGF-β1 versus TGF-β1 group, n = 3 per group).
Immunofluorescence staining showed an increased average immu￾nofluorescence intensity of FN, α-SMA and F-actin in RTFs after TGF-β1
treatment (Fig. 3A–G; p < 0.001 in FN, p < 0.01 in α-SMA or p < 0.05 in
F-actin versus DMSO group; n = 3), and these fibrotic responses were
significantly suppressed by FDI-6 treatment (Fig. 3A–G; p˂0.001 in FN, p
< 0.001 in α-SMA or p < 0.05 in F-actin versus TGF-β1 group; n = 3). It
has been reported that dysregulation of TGF-β1/Smad pathway is an
important pathogenic mechanism in tissue fibrosis. Smad2 and Smad3
are the two major downstream regulators which promote TGF-β1-
mediated tissue fibrosis (Hu et al., 2018). The phosphorylation levels of
Smad2/3 were examined in RTFs after FDI-6 treatment. As shown in
Fig. 4C, FDI-6 significantly decreased the phosphorylation levels of
Smad2/3 in RTFs, regardless of the existence of TGF-β1 (p < 0.001 in
FDI-6 and FDI-6+TGF-β1 group versus DMSO and TGF-β1 group
respectively; n = 3 per group). Together, these results showed that FDI-6
regulated the expression of these profibrotic genes in RTFs likely
through the TGF-β1/Smad2/3 signaling pathway.
3.5. FDI-6 was safe for subconjunctival injection in rabbit eyes after
trabeculectomy
Subconjunctival fibrosis after trabeculectomy may cause a failure of
filtering surgeries. Therefore, the antifibrotic efficacy of FDI-6 in the
maintenance of functional bleb in a rabbit trabeculectomy model was
studied. Fig. 5A shows the overall animal experimental design. Briefly,
20 rabbit eyes were assigned into the non-surgical groups including the
blank control, saline only and FDI-6 only groups, and the surgical groups
including the surgery + saline group and the surgery + FDI-6 group.
Based on corneal fluorescence staining analysis (Fig. 5B), there was no
positive corneal fluorescence staining in all rabbit eyes, suggesting no
Fig. 1. FDI-6 inhibited expression of FOXM1 protein and cell proliferation in cultured RTFs. (A)Western blot and quantification of FOXM1 expression in RTFs treated
with different concentrations of FDI-6 (2.5, 5, 10, 20, 30 and 40 μM). GAPDH was used as a reference protein, and these values were further normalized to that value
of DMSO group. *P < 0.05, **P < 0.01 and ***P < 0.001 versus DMSO group by Tukey post hoc test (n = 3 per group). (B) Cell proliferative viability under different
FDI-6 concentrations (2.5, 5, 10, 20 and 40 μM). ††P < 0.01 and †††P < 0.001 versus blank control at 24 h by Tamhane’s T2 post hoc test (n = 3 per group). (C)
Correspondingly, cell number was counted under a light microscope at × 200 magnification. †P < 0.05, ††P < 0.01 and †††P < 0.001 versus blank control at 24 h by
Tamhane’s T2 post hoc test (n = 3 per group), ##P < 0.01 and ###P < 0.001 at 24 h versus that at 0 h by paired t-test (n = 3 per group). Results were shown as mean
SEM.
C. Lan et al.
Experimental Eye Research 210 (2021) 108725
6 adverse or toxic effects on ocular surface after FDI-6 treatment. No
obvious complications were observed in all groups within the 14-
d period, such as corneal or conjunctival epithelial defects, bleb leaks,
hypotony, cataract and endophthalmitis when assessed with a slit-lamp
microscope (Fig. 5B). No corneal or conjunctival TUNEL positive cells
were found in FDI-6 only group (Fig. 5C and D). Conjunctival hyperemia
was mild in the surgery + saline group and the surgery + FDI-6 group
and lasted for approximately 1 week. There was no remarkable
difference between the surgery + saline group and the surgery + FDI-6
group in general ocular examination, except the scar formation.
3.6. FDI-6 promoted functional bleb formation in rabbits after
trabeculectomy
The postoperative bleb status was graded according to Krofeld et al.
(Hu et al., 2018). As showed in Fig. 6B, the surgery with FDI-6 group had
Fig. 2. FDI-6 reduced the TGF-β1-induced cell migration and proliferation, and attenuated the TGF-β1-induced cell contraction in cultured RTFs. The wound healing
assay and CCK-8 assay were performed in blank control, DMSO, TGF-β1 (20 ng/ml), FDI-6 (10 μM) + TGF-β1 (20 ng/ml), and FDI-6 (10 μM) conditions. The groups
were observed for 24 h. (A, B) Representative images and statistical results of scratch wound healing migration in RTFs at 0 h, 12 h, and 24 h after treatment. Edges of
the migrated cells were dotted with red lines. (C) Cell proliferation of RTFs was determined in the five groups at 24 h. (D) Representative images and quantitative
analysis of gel contraction of RTFs in the five groups at 0 h, 12 h and 24 h after treatment. The percentage (%) of contraction area was calculated according to the
ratio of contracted area comparing to the initial area. (E) Phase-contrast microscopy images of cellular morphology at 0 h and 12 h post-treatment. Dotted red box:
enlarged images of details. One-way ANOVA followed by Tamhane’s T2 post hoc test was used to test the variations among groups (n = 3 per group). *P < 0.05, **P
< 0.01 and ***P < 0.001 versus TGF-β1 group, #P < 0.05, ##P < 0.01 and ###P < 0.001 versus DMSO group. Results were shown as mean ± SEM. Scale bars: 300 μm
in (A) and 200 μm in (E). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
C. Lan et al.
Experimental Eye Research 210 (2021) 108725
7 a better score at DPO 14 compared with the control group (p < 0.05; n =
4 per group). Corresponding to the results of bleb score, the blebs
appeared to be small, flat and abundantly vascularized in the surgery +
saline group at DPO 7 and DPO 14 and were changed to be slightly
bulged and less vascularized in the surgery + FDI-6 group (Fig. 6A).
In Fig. 6C, preoperative IOP values showed no significant difference
among all groups, and IOP at DPO 1 to DPO 14 did not differ among the
non-surgery groups (p > 0.05; n = 4 per group). IOP in the surgical
groups showed a significant decrease at DPO 1 to DPO 4 and a gradual
elevation at DPO 5 to DPO 14. Moreover, IOP in the surgery + FDI-6
group (9 ± 0.74 and 8.58 ± 0.51 mmHg at DPO 12 and DPO 14,
respectively) was significantly lower than that in the surgery + saline
group (10.42 ± 1.62 and 9.75 ± 1.14 mmHg at DPO 12 and DPO 14,
respectively) at the two time points (p < 0.05, n = 4 per group). After
Fig. 3. Immunofluorescence of fibronectin, α-SMA and F-actin in RTFs. Each column from left to right represented the result in blank control, DMSO, TGF-β1 (20 ng/
ml), FDI-6 (10 μM) + TGF-β1 (20 ng/ml), and FDI-6 (10 μM) groups, and each group was generally observed for 24 h. In each panel, the first row showed cells stained
with DAPI, and the second row showed expression levels of fibronectin (panel A), α-SMA (panel B) and F-actin (panel C; yellow: residual FDI-6 particles). Boxed
regions were enlarged at the upper right corner. Scale bars: 100 μm. Statistical results of the mean Fibronectin (D), α-SMA (E) and F-actin (F) fluorescent intensity.
One-way ANOVA followed by Tukey post hoc test was used to test the variations among groups (n = 3 per group). *P < 0.05 and ***P < 0.001 versus TGF-β1 group, #P < 0.05, ##P < 0.01 and ###P < 0.001 versus DMSO group. Results were shown as mean ± SEM. (For interpretation of the references to colour in this figure legend,
the reader is referred to the Web version of this article.)
C. Lan et al.
Experimental Eye Research 210 (2021) 108725
8 DPO 12, IOP in the surgery + saline group reached near to the level of
the saline only group (10.79 ± 0.78 mmHg, p > 0.05), while IOP in the
surgery + FDI-6 group was still significantly lower than that in FDI-6
only group(p < 0.05). This indicates that FDI-6 could maintain the
decreased IOP for a period of time after trabeculectomy.
As shown in Fig. 6D, all rabbits in non-surgical groups showed an
intact conjunctiva epithelium and loose subepithelial connective tissue
spaces. The conjunctival space (superficial and deep stroma) in the
surgery + saline group was changed into a compact and optically dense
matrix and, consequently, lack of filtering space for aqueous outflow,
which was attenuated in the surgery + FDI-6 group. These results sug￾gested that FDI-6 treatment reduced subconjunctival fibrosis and further
promoted functional bleb formation. In addition, a high level of
infiltration of inflammatory cells showing a bright white, circular or
elliptical hyper-reflective morphology (black arrows, presumably
derived from lymphocyte and granulocyte) was observed in the
conjunctival epithelium of the surgery + saline group, and decreased in
the surgery + FDI-6 group, indicating a potential anti-inflammatory
effect of FDI-6.
3.7. FDI-6 attenuated subconjunctival fibrosis in rabbits after
trabeculectomy
To evaluate the subconjunctival fibrotic response to trabeculectomy￾induced injury, expressions of FOXM1, α-SMA, COL1A1 and FN in bleb
tissue were analyzed. As showed in Fig. 7E, the levels of these proteins
Fig. 4. FDI-6 downregulated the TGF-β1-induced the expression of profibrotic genes in cultured RTFs. (A, C) Representative Western blot images and quantified
expression levels of FOXM1, FN, COL1A1, α-SMA, p-Smad2/3 and total Smad3 at 24 h post-treatment. (n = 3 per group). GAPDH was used as a reference protein, and
these values were further normalized to that value of DMSO group. Phosphorylation levels of Smad2/3 were quantified as the fold value of p-Smad2/3 and total
Smad3. (B) The mRNA expression analysis of α-SMA, COL1A1 and FN at 24 h after treatment (n = 4 per group). One-way ANOVA was used to test the variations
among groups in both Western blot and mRNA analysis by Tamhane’s T2 post hoc test. **P < 0.01 and ***P < 0.001 versus TGF-β1 group; #P < 0.05, ##P < 0.01 and ###P < 0.001 versus DMSO group. Results were shown as mean ± SEM.
C. Lan et al.
Experimental Eye Research 210 (2021) 108725
9 significantly elevated after trabeculectomy, indicating that trabeculec￾tomy activated the expression of profibrotic genes, while FDI-6 reduced
the expression of these genes. Consistent with the immunoblot findings,
H&E staining showed an increased thickness between conjunctiva and
sclera in the surgical groups (Fig. 7A and C; p < 0.05 versus no surgery
group; n = 3 per group). The mean thickness in the surgery + FDI-6
group was tended to be thinner than that in the surgery + saline group,
although the intergroup difference was not statistically significant
(Fig. 7A and C; p > 0.05; n = 3). Masson’s trichrome staining demon￾strated that substantial number of collagen accumulated in
subconjunctival area in the surgical groups (Fig. 7A and D, p < 0.05 in
surgery + FDI-6 group or p < 0.001 in surgery + saline group versus
saline only group; n = 3 per group), and collagen volume fraction in the
surgery + FDI-6 group (72.8%) was significantly lower than that in the
surgery + saline group (85.8%) (Fig. 7A and D, p < 0.05; n = 3). In
addition, immunohistochemistry of α-SMA between the conjunctiva and
the sclera showed positive expression in the surgical groups and nega￾tive expression in the non-surgical groups. The FDI-6+surgery group
showed a weaker α-SMA expression than the surgery + saline group,
suggesting the presence of fewer myofibroblasts (Fig. 7A).
Fig. 5. Postoperative examinations of FDI-6-treated anterior segment. (A) Experimental flow scheme for animals after trabeculectomy. Rabbits were divided into five
groups, including the blank control, saline only, FDI-6 (40 μM) only, surgery + saline and surgery + FDI-6 (40 μM) groups. (B) Representative images of corneal
fluorescence staining and anterior segments at DPO 14. (C, D) Representative images of TUNEL staining in the surrounding tissues of surgery constructed filtering
areas (panel C) and corneas (panel D). Blue, DAPI. Scale bars: 200 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the
Web version of this article.)
C. Lan et al.
Experimental Eye Research 210 (2021) 108725
10 Immunofluorescence data of COL1A1 displayed the same trend as
immunohistochemistry of α-SMA (Fig. 7B). Taken together, our data
indicated that FDI-6 prevented subconjunctival fibrosis after trabecu￾lectomy by reducing myofibroblast differentiation and further expres￾sion of ECM including FN and COL1A1 in rabbits.
4. Discussion
To date, applications of intraoperative and postoperative antifibrotic
drugs, such as MMC and 5-FU, have been limited by their side effects.
Novel antifibrotic agents such as vascular endothelial growth factor
inhibitors (Van Bergen et al., 2011), TGF-β1 (Yamanaka et al., 2006),
Fig. 6. FDI-6 promoted functional bleb formation and maintained the decreased IOP for a period of time in rabbits after trabeculectomy. (A) Representative
morphological images of filtering bleb at DPO 7 and DPO 14 among all groups. (B) Statistical analysis of bleb scoring between the surgery + FDI-6 (40 μM) group and
the surgery + saline group. (C) IOPs of the operated and non-operated eyes at different time points. The DPO indicates the postoperative day. (D) In vivo confocal
images of conjunctival epithelium, superficial stroma and deep stroma in the area of filtering bleb at DPO 14. Arrowheads indicate inflammatory cells infiltration.
Paired Student’s t-test was used in Fig. 5B to calculate statistical differences (n = 4 per group). One-way ANOVA followed by Tamhane’s T2 post hoc test was used in
Fig. 5C to test the variations among groups (n = 4 per group). *P < 0.05 versus surgery + saline group. Results were shown as mean ± SEM.
C. Lan et al.
Experimental Eye Research 210 (2021) 108725
11 matrix metalloproteinases (MMPs) (Liu et al., 2004), microRNA (Ran
et al., 2015), rosiglitazone (Zhang et al., 2019) and losartan (Shi et al.,
2017) have been emerging at a rapid rate, but few have been translated
into clinical usage. In this study, we investigated the effect and under￾lying mechanism of FDI-6 on subconjunctival fibrosis and found that
FDI-6 was an effective pharmacologic inhibition of FOXM1 to prevent
subconjunctival fibrosis and increase the success rate of trabeculectomy.
As mentioned in the introduction, FOXM1 plays an important role in
fibrotic disease, and FOXM1 inhibition down-regulates the fibrosis￾related gene expression and attenuates fibrosis-associated phenotype
(Goda et al., 2020; Jeng et al., 2020; Katoh, 2018; Yang et al., 2016).
FOXM1 is highly expressed in lung fibroblasts isolated from idiopathic
pulmonary fibrosis (IPF) patients. Siomycin A (Sio A), an inhibitor of
FOXM1 binding to DNA, significantly attenuated myofibroblasts and
reduced expression of profibrotic genes (Yang et al., 2016). Moreover, a
small interfering RNA resulted in FOXM1 silencing, inhibited the acti￾vation of keloid fibroblasts and extracellular matrix production by
down-regulating the gene expression through the TGF-β1/Smad
pathway (Lam et al., 2013). Our results, consistent with these studies,
showed FDI-6 significantly reduced expression of FOXM1 and ECM
proteins including α-SMA, COL1A1 and FN in the cultured RTFs, and
inhibited its proliferation, migration and contraction. In addition, the
mean fluorescence intensity of FN, α-SMA and F-actin was weakened in
cultured RTFs treated with FDI-6, indicating a reduced ECM synthesis.
Previous studies showed that the obstruction of artificial outflow
channel was mainly caused by the excessive ECM deposition and
contraction after trabeculectomy, ultimately causing IOP elevation fol￾lowed by surgical failure (Darby et al., 2007; Igarashi et al., 2021). In the
current study, in vivo confocal microscopic analysis revealed that the
trabeculectomy induced dense fibers in both the superficial and deep
Fig. 7. FDI-6 attenuated subconjunctival fibrosis in rabbits after trabeculectomy. (A) Representative histological images of the operative area. H&E staining (top
row), Masson’ trichrome staining (middle row) and α-SMA immunohistochemistry (bottom row) were conducted respectively. Arrowheads indicate the area of
filtration channels. c: conjunctiva, s: sclera, r: ciliary body. Scale bars: 200 μM. (B) Representative immunofluorescent images for specific marker, including COL1
(red) and DAPI (blue), in the surrounding tissues of surgery constructed filtering areas. Box in bottom left was a magnified view. Scale bars: 200 μM. (C) Mean
thickness between the conjunctiva and sclera in the area of filtering bleb. (D) Collagen volume fraction was calculated in Masson’ trichrome staining. (E) Western blot
images of FOXM1, FN, COL1A1 and α-SMA at DPO 14. One-way ANOVA followed by Tamhane’s T2 post hoc test was used to test the variations among groups (n = 3
per group). *P < 0.05 versus surgery + saline group; #P < 0.05, ##P < 0.01 and ###P < 0.001 versus saline only or FDI-6 only group. Results were shown as mean ±
SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
C. Lan et al.
Experimental Eye Research 210 (2021) 108725
12 stroma layers were decreased in rabbit eyes by FDI-6 subconjunctival
injection. More importantly, histological analysis of rabbit eyes also
showed a decreased subconjunctival collagen deposition and a
down-regulated expression of fibrotic genes including α-SMA and
COL1A1 in the FDI-6 treatment group. The decreased scaring formation
of surgical site of rabbit eyes induced by FDI-6 treatment could further
improve the filtering bleb function. These findings were consistent with
our current results in RTFs, in which FDI-6 could attenuate the fibrotic
response induced by TGF-β, suggesting that FDI-6 exerts anti-fibrotic
effects mainly by down-regulating the proliferation, migration and
contraction of RTFs in a rabbit trabeculectomy model.
Chronic ocular hypertension animal models and/or glaucoma pa￾tients were recruited in previous studies to investigate the potential ef￾fect of MMC (Al Habash et al., 2015), 5-FU (Green et al., 2014) and
Rosiglitazone (Zhang et al., 2019). These studies have showed these
medications have an early significant efficacy on IOP control after tra￾beculectomy. Our study showed that FDI-6 exerts a significant efficacy
against IOP elevation in healthy rabbits at DPO 12 and 14. This differ￾ence may be attributed to the different IOP baselines of different in￾dividuals before treatment of medications. The IOP was monitored every
other day for 14 days in this study since the peak of subconjunctival
fibrosis following trabeculectomy usually occurs at DPO 7 (Miller et al.,
1989; Doyle et al., 1993). Even so, we still found a significant efficacy of
FDI-6 on IOP control at DPO 14. It is speculated that FDI-6 treatment
might maintain the decreased IOP for a certain period of time post DPO
14. Consistent with the results in postoperative IOP, the bleb formation
and subconjunctival fiber density following trabeculectomy were
improved by FDI-6 treatment at DPO 14, indicating an antifibrotic effect
of FDI-6.
Previous studies demonstrated that FOXM1 also played a crucial role
in the progress of inflammation in different tissues and organs, such as
lung (Balli et al., 2012; Ren et al., 2013), prostate (Pan et al., 2018),
bone (Zeng et al., 2019) and liver (Kurahashi et al., 2020), where the
inflammatory reaction was attenuated by inhibition of FOXM1 with
pharmacology or biological methods. It was known that the inflamma￾tory phase was an important part of pathobiology of subconjunctival
wound healing (Yamanaka et al., 2015), and specific inflammatory cells
and their chemical mediators were critical to promoting multiple tissue
or organ fibrosis (Coentro et al., 2019; Marcelin et al., 2019; Tang et al.,
2019). Interestingly, based on the confocal microscopy, we found that a
high level of inflammatory cells was infiltrated into the conjunctival
epithelium on Day 14 after trabeculectomy, whose level was decreased
after FDI-6 treatment. Thus, it remains to be confirmed whether FDI-6
exerts a similar anti-inflammation effect on the eye after
trabeculectomy.
Antimetabolites including MMC and 5-FU, which are commonly used
in the prevention and treatment of scarring following glaucoma filtra￾tion surgery (GFS), play an anti-proliferative and fibrotic role mainly by
inhibiting DNA syntheses, inducing apoptosis and blocking cell prolif￾eration. These effects cause significant toxicity to the cells, causing
dysfunction of the corneal epithelium, hypotony, bleb leakage or bleb￾related complications (Al Habash et al., 2015; Green et al., 2014).
However, previous studies showed that the inhibition of FOXM1 is the
main mechanism underlying the antifibrotic effect of FDI-6. This is
consistent with our results showing that FDI-6 inhibited transcription of
FOXM1 regulated fibrotic genes by blocking the FOXM1 binding site on
chromatin, instead of causing cellular toxicities. As shown in Figs. 5–7,
our results demonstrated that the FDI-6-treated rabbit eyes showed
similar results as blank and saline-treated eyes, suggesting that the 40
μM of FDI-6 had no toxic effect on normal ocular tissues of rabbits. After
trabeculectomy, conjunctival hyperemia was mild and lasted for
approximately 1 week in the rabbit eyes. No corneal edema or other
corneal or conjunctival disorders was detected in any group. No obvious
inflammatory signs in the anterior chamber of the eye were observed in
all the rabbits. The confocal microscopic and histological analysis
revealed the intact architecture of conjunctival epithelium in all groups.
Additionally, no TUNEL-positive cells were observed in the cornea or
conjunctiva of the FDI-6 treated eyes, indicating that FDI-6 appeared to
be safe as an antiproliferative medication used on the trabeculectomy
rabbit model. As a limitation of this research, the experiments were
conducted for only 14 days, and therefore longer observations are
needed to investigate the antifibrotic effects of FDI-6 in filtering sur￾geries of glaucoma as well as its other potential side effects.
In summary, our study showed that FDI-6 inhibited the TGF-β1-
induced ECM proteins synthesis, myofibroblast activation, and cellular
migration, proliferation and collagen gel contraction in cultured RTFs.
This appears to occur, at least in part, via blocking the TGF-β1/Smad2/3
signaling pathway. Furthermore, FDI-6 represents a safe and efficient
reagent capable of attenuating subconjunctival fibrosis and promoting
functional bleb formation after trabeculectomy. Further studies focusing
on optimization of the concentration and timing of FDI-6 delivery, and
longer observation of its antifibrotic effects and side effects, should be
needed.
Author contributions
Y. Z, X. L and N. F conceived and designed the experiments, and
supervised the study. C. L performed experiments, analyzed the data,
and wrote the manuscript. J. T, G. L, L. T, L. H, XL. L and L. Z were
involved in conducting of the animal experiments, and/or participated
in analysis of data and manuscript editing. All authors read and dis￾cussed the results, and reviewed the final version of the manuscript.
Graphical abstract. Subconjunctival fibrosis occurred after trabe￾culectomy, with myofibroblasts trans-differentiation and further up￾regulation of profibrotic genes including α-SMA, Collagen I and Fibro￾nectin. FDI-6 inhibited transcription of FOXM1 regulated genes with
FOXM1 displaced from chromatin, and then resulted in a decreased
expression of these profibrotic genes through blocking the TGF-β1/
Smad2/3 signaling pathway, ultimately attenuating subconjunctival
fibrosis.
Declaration of competing interest
All authors declare no competing interests.
Acknowledgments
This study was supported by the National Natural Science Founda￾tion of China (NSFC Grant Nos. 81770924, 81900829, 81500718 and
81970789), Sanming Project of Medicine in Shenzhen (No.
SZSM201512045), the Science and Technology Plan Project in Medical
and Health of Xiamen (Grants Nos. 3502Z20194066 and
3502Z20194069), Science and Technology Innovation Committee of
Shenzhen (No. JCYJ20190807153005579) and Guangdong Basic and
Applied Basic Research Foundation (Grants No. 2019A1515011234).
References
Al Habash, A., Aljasim, L.A., Owaidhah, O., Edward, D.P., 2015. A review of the efficacy
of mitomycin C in glaucoma filtration surgery. Clin. Ophthalmol. 9, 1945–1951.

https://doi.org/10.2147/OPTH.S80111.

Balli, D., Ren, X., Chou, F.S., Cross, E., Zhang, Y., Kalinichenko, V.V., Kalin, T.V., 2012.
Foxm1 transcription factor is required for macrophage migration during lung
inflammation and tumor formation. Oncogene 31, 3875–3888. https://doi.org/
10.1038/onc.2011.549.
Bell, K., de Padua Soares Bezerra, B., Mofokeng, M., Montesano, G., Nongpiur, M.E.,
Marti, M.V., Lawlor, M., 2020. Learning from the past: mitomycin C use in
trabeculectomy and its application in bleb-forming minimally invasive glaucoma
surgery. Surv. Ophthalmol. 66, 109–123.
Cabourne, E., Clarke, J.C., Schlottmann, P.G., Evans, J.R., 2015. Mitomycin C versus 5-
Fluorouracil for wound healing in glaucoma surgery. Cochrane Database Syst. Rev.
CD006259 https://doi.org/10.1002/14651858.CD006259.pub2.
Chen, K., Lai, K., Zhang, X., Qin, Z., Fu, Q., Luo, C., Jin, X., Hu, J., Liu, S., Yao, K., 2019.
Bromfenac inhibits TGF-beta1-induced fibrotic effects in human pterygium and
conjunctival fibroblasts. Invest. Ophthalmol. Vis. Sci. 60, 1156–1164. https://doi.
org/10.1167/iovs.18-24743.
C. Lan et al.
Experimental Eye Research 210 (2021) 108725
13
Coentro, J.Q., Pugliese, E., Hanley, G., Raghunath, M., Zeugolis, D.I., 2019. Current and
upcoming therapies to modulate skin scarring and fibrosis. Adv. Drug Deliv. Rev.
146, 37–59. https://doi.org/10.1016/j.addr.2018.08.009.
Davis, B.M., Crawley, L., Pahlitzsch, M., Javaid, F., Cordeiro, M.F., 2016. Glaucoma: the
retina and beyond. Acta Neuropathol. 132, 807–826. https://doi.org/10.1007/
s00401-016-1609-2.
Doyle, J.W., Sherwood, M.B., Khaw, P.T., McGrory, S., Smith, M.F., 1993. Intraoperative
5-fluorouracil for filtration surgery in the rabbit. Invest. Ophthalmol. Vis. Sci. 34,
3313–3319. https://doi.org/10.1007/BF00921967.
Darby, I.A., Hewitson, T.D., 2007. Fibroblast differentiation in wound healing and
fibrosis. Int. Rev. Cytol. 257, 143–179. https://doi.org/10.1016/S0074-7696(07)
57004-X.Gedde.
Gedde, S.J., Feuer, W.J., Shi, W., Lim, K.S., Barton, K., Goyal, S., Ahmed, I.I.K.,
Brandt, J., 2018. Treatment outcomes in the primary tube versus trabeculectomy
study after 1 Year of follow-up. Ophthalmology 125, 650–663. https://doi.org/
10.1016/j.ophtha.2018.02.003.
Goda, C., Balli, D., Black, M., Milewski, D., Le, T., Ustiyan, V., Ren, X., Kalinichenko, V.
V., Kalin, T.V., 2020. Loss of FOXM1 in macrophages promotes pulmonary fibrosis
by activating p38 MAPK signaling pathway. PLoS Genet. 16, e1008692 https://doi.
org/10.1371/journal.pgen.1008692.
Gormally, M.V., Dexheimer, T.S., Marsico, G., Sanders, D.A., Lowe, C., Matak￾Vinkovi´c, D., Michael, S., Jadhav, A., Rai, G., Maloney, D.J., Simeonov, A.,
Balasubramanian, S., 2014. Suppression of the FOXM1 transcriptional programme
via novel small molecule inhibition. Nat. Commun. 5, 5165. https://doi.org/
10.1038/ncomms6165.
Green, E., Wilkins, M., Bunce, C., Wormald, R., 2014. 5-Fluorouracil for glaucoma
surgery. Cochrane. Database Syst. Rev. CD001132 https://doi.org/10.1002/
14651858.CD001132.pub2.
Halasi, M., Gartel, A.L., 2013. FOX(M1) news–it is cancer. Mol. Canc. Therapeut. 12,
245–254. https://doi.org/10.1158/1535-7163.MCT-12-0712.
Han, F., Shu, J., Wang, S., Tang, C., Luo, F., 2019. Metformin inhibits the expression of
biomarkers of fibrosis of EPCs in vitro. Stem Cell. Int. 2019, 9019648. https://doi.
org/10.1155/2019/9019648.
Higginbotham, E.J., Stevens, R.K., Musch, D.C., Karp, K.O., Lichter, P.R., Bergstrom, T.J.,
Skuta, G.L., 1996. Bleb-related endophthalmitis after trabeculectomy with
mitomycin C. Ophthalmology 103, 650–656. https://doi.org/10.1016/s0161-6420
(96)30639-8.
Hu, H., Chen, D., Wang, Y., Feng, Y., Cao, G., Vaziri, N.D., Zhao, Y., 2018. New insights
into TGF-beta/Smad signaling in tissue fibrosis. Chem. Biol. Interact. 292, 76–83.

https://doi.org/10.1016/j.cbi.2018.07.008.

Igarashi, N., Honjo, M., Aihara, M., 2021. Effects of mammalian target of rapamycin
inhibitors on fibrosis after trabeculectomy. Exp. Eye Res. 203, 108421. https://doi.
org/10.1016/j.exer.2020.108421.
Jeng, K.-S., Lu, S.-J., Wang, C.-H., Chang, C.-F., 2020. Liver fibrosis and inflammation
under the control of ERK2. Int. J. Mol. Sci. 21 https://doi.org/10.3390/
ijms21113796.
Kang, Jessica Minjy, Tanna Angelo, P., 2021. Glaucoma. Med. Clin. 105, 493–510.

https://doi.org/10.1016/j.mcna.2021.01.004.

Kalinichenko, V.V., Kalin, T.V., 2015. Is there potential to target FOXM1 for
’undruggable’ lung cancers? Expert Opin. Ther. Targets 19, 865–867. https://doi.
org/10.1517/14728222.2015.1042366.
Katoh, M., 2018. Multilayered prevention and treatment of chronic inflammation, organ
fibrosis and cancer associated with canonical WNT/betacatenin signaling activation
(Review). Int. J. Mol. Med. 42, 713–725. https://doi.org/10.3892/ijmm.2018.3689.
Klingberg, F., Hinz, B., White, E.S., 2013. The myofibroblast matrix: implications for
tissue repair and fibrosis. J. Pathol. 229, 298–309. https://doi.org/10.1002/
path.4104.
Koo, C.Y., Muir, K.W., Lam, E.W., 2012. FOXM1: from cancer initiation to progression
and treatment. Biochim. Biophys. Acta 1819, 28–37. https://doi.org/10.1016/j.
bbagrm.2011.09.004.
Kurahashi, T., Yoshida, Y., Ogura, S., Egawa, M., Furuta, K., Hikita, H., Kodama, T.,
Sakamori, R., Kiso, S., Kamada, Y., Wang, I.C., Eguchi, H., Morii, E., Doki, Y.,
Mori, M., Kalinichenko, V.V., Tatsumi, T., Takehara, T., 2020. Forkhead box M1
transcription factor drives liver inflammation linking to hepatocarcinogenesis in
mice. Cell Mol. Gastroenterol Hepatol. 9, 425–446. https://doi.org/10.1016/j.
jcmgh.2019.10.008.
Lam, E.W., Brosens, J.J., Gomes, A.R., Koo, C.Y., 2013. Forkhead box proteins: tuning
forks for transcriptional harmony. Nat. Rev. Canc. 13, 482–495. https://doi.org/
10.1038/nrc3539.
Lama, P.J., Fechtner, R.D., 2003. Antifibrotics and wound healing in glaucoma surgery.
Surv. Ophthalmol. 48, 314–346. https://doi.org/10.1016/s0039-6257(03)00038-9.
Li, Y., Lu, L., Tu, J., Zhang, J., Xiong, T., Fan, W., Wang, J., Li, M., Chen, Y., Steggerda, J.,
Peng, H., Chen, Y., Li, T.W.H., Zhou, Z., Mato, J.M., Seki, E., Liu, T., Yang, H., Lu, S.,
2020. Reciprocal regulation between forkhead box M1/NF-kappaB and methionine
adenosyltransferase 1A drives liver cancer. Hepatology 72, 1682–1700. https://doi.
org/10.1002/hep.31196.
Liu, C., Huang, Y., Chiu, A.W., Ju, J., 2004. Transcript expression of matrix
metalloproteinases in the conjunctiva following glaucoma filtration surgery in
rabbits. Ophthalmic Res. 36, 114–119. https://doi.org/10.1159/000076891.
Liu, Y., Ko, J.A., Yanai, R., Kimura, K., Chikama, T., Sagara, T., Nishida, T., 2008.
Induction by latanoprost of collagen gel contraction mediated by human tenon
fibroblasts: role of intracellular signaling molecules. Invest. Ophthalmol. Vis. Sci. 49,
1429–1436. https://doi.org/10.1167/iovs.07-0451.
Lusthaus, J., Goldberg, I., 2019. Current management of glaucoma. Med. J. Aust. 210,
180–187. https://doi.org/10.5694/mja2.50020.
Marcelin, G., Silveira, A.L.M., Martins, L.B., Ferreira, A.V., Clement, K., 2019.
Deciphering the cellular interplays underlying obesity-induced adipose tissue
fibrosis. J. Clin. Invest. 129, 4032–4040. https://doi.org/10.1172/JCI129192.
Mearza, A.A., Aslanides, I.M., 2007. Uses and complications of mitomycin C in
ophthalmology. Expet Opin. Drug Saf. 6, 27–32. https://doi.org/10.1517/
14740338.6.1.27.
Miller, M.H., Grierson, I., Unger, W.I., Hitchings, R.A., 1989. Wound healing in an animal
model of glaucoma fistulizing surgery in the rabbit. Ophthalmic Surg. 20, 350–357.

https://doi.org/10.1055/s-2008-1046393.

Nacu, N., Luzina, I.G., Highsmith, K., Lockatell, V., Pochetuhen, K., Cooper, Z.A.,
Gillmeister, M.P., Todd, N.W., Atamas, S.P., 2008. Macrophages produce TGF-beta￾induced (beta-ig-h3) following ingestion of apoptotic cells and regulate MMP14
levels and collagen turnover in fibroblasts. J. Immunol. 180, 5036–5044. https://
doi.org/10.4049/jimmunol.180.7.5036.
Ngu, J.M., Teng, G., Meijndert, H.C., Mewhort, H.E., Turnbull, J.D., Stetler￾Stevenson, W.G., Fedak, P.W., 2014. Human cardiac fibroblast extracellular matrix
remodeling: dual effects of tissue inhibitor of metalloproteinase-2. Cardiovasc.
Pathol. 23, 335–343. https://doi.org/10.1016/j.carpath.2014.06.003.
NikhalaShree, S., Karthikkeyan, G., George, R., Shantha, B., Vijaya, L., Ratra, V.,
Sulochana, K.N., Coral, K., 2019. Lowered decorin with aberrant extracellular matrix
remodeling in aqueous humor and tenon’s tissue from primary glaucoma patients.
Invest. Ophthalmol. Vis. Sci. 60, 4661–4669. https://doi.org/10.1167/iovs.19-
27091.
Pan, C., Chen, Y., Xu, T., Wang, J., Li, D., Han, X., 2018. Chronic exposure to microcystin￾leucine-arginine promoted proliferation of prostate epithelial cells resulting in
benign prostatic hyperplasia. Environ. Pollut. 242, 1535–1545. https://doi.org/
10.1016/j.envpol.2018.08.024.
Paul, C., 2000. The Advanced Glaucoma Intervention Study (AGIS): 7. The relationship
between control of intraocular pressure and visual field deterioration. The AGIS
Investigators. Am. J. Ophthalmol. 130, 429–440. https://doi.org/10.1016/s0002-
9394(00)00538-9.
Perkins, T.W., Faha, B., Ni, M., Kiland, J.A., Poulsen, G.L., Antelman, D., Atencio, I.,
Shinoda, J., Sinha, D., Brumback, L., Maneval, D., Kaufman, P.L., Nickells, R.W.,
2002. Adenovirus-mediated gene therapy using human p21WAF-1/Cip-1 to prevent
wound healing in a rabbit model of glaucoma filtration surgery. Arch. Ophthalmol.
120, 941–949. https://doi.org/10.1001/archopht.120.7.941.
Quigley, H.A., Broman, A.T., 2006. The number of people with glaucoma worldwide in
2010 and 2020. Br. J. Ophthalmol. 90, 262–267. https://doi.org/10.1136/
bjo.2005.081224.
Ran, W., Zhu, D., Feng, Q., 2015. TGF-beta2 stimulates Tenon’s capsule fibroblast
proliferation in patients with glaucoma via suppression of miR-29b expression
regulated by Nrf2. Int. J. Clin. Exp. Pathol. 8, 4799–4806. https://pubmed.ncbi.nlm.
nih.gov/26191170.
Ren, X., Shah, T.A., Ustiyan, V., Zhang, Y., Shinn, J., Chen, G., Whitsett, J.A., Kalin, T.V.,
Kalinichenko, V.V., 2013. FOXM1 promotes allergen-induced goblet cell metaplasia
and pulmonary inflammation. Mol. Cell Biol. 33, 371–386. https://doi.org/10.1128/
MCB.00934-12.
Santos, J.M., Camoes, ˜ S.P., Filipe, E., Cipriano, M., Barcia, R.N., Filipe, M., Teixeira, M.,
Simoes, ˜ S., Gaspar, M., Mosqueira, D., Nascimento, D.S., Pinto-do-O, ´ P., Cruz, P.,
Cruz, H., Castro, M., Miranda, J.P., 2015. Three-dimensional spheroid cell culture of
umbilical cord tissue-derived mesenchymal stromal cells leads to enhanced
paracrine induction of wound healing. Stem Cell Res. Ther. 6, 90. https://doi.org/
10.1186/s13287-015-0082-5.
Schlunck, G., Meyer-ter-Vehn, T., Klink, T., Grehn, F., 2016. Conjunctival fibrosis
following filtering glaucoma surgery. Exp. Eye Res. 142, 76–82. https://doi.org/
10.1016/j.exer.2015.03.021.
Shi, H., Wang, H., Fu, S., Xu, K., Zhang, X., Xiao, Y., Ye, W., 2017. Losartan attenuates
scar formation in filtering bleb after trabeculectomy. Invest. Ophthalmol. Vis. Sci.
58, 1478–1486. https://doi.org/10.1167/iovs.16-21163.
Shu, D., Lovicu, F.J., 2017. Myofibroblast transdifferentiation: the dark force in ocular
wound healing and fibrosis. Prog. Retin. Eye Res. 60, 44–65. https://doi.org/
10.1016/j.preteyeres.2017.08.001.
Tan, J., Liu, G., Zhu, X., Wu, Z., Wang, N., Zhou, L., Zhang, X., Fan, N., Liu, X., 2019.
Lentiviral vector-mediated expression of exoenzyme C3 transferase lowers
intraocular pressure in monkeys. Mol. Ther. 27, 1327–1338. https://doi.org/
10.1016/j.ymthe.2019.04.021.
Tan, J., Wang, X., Cai, S., He, F., Zhang, D., Li, D., Zhu, X., Zhou, L., Fan, N., Liu, X.,
2020. C3 transferase-expressing scAAV2 transduces ocular anterior segment tissues
and lowers intraocular pressure in mouse and monkey. Mol. Ther. Methods Clin.
Dev. 17, 143–155. https://doi.org/10.1016/j.omtm.2019.11.017.
Tang, P., Nikolic-Paterson, D.J., Lan, H., 2019. Macrophages: versatile players in renal
inflammation and fibrosis. Nat. Rev. Nephrol. 15, 144–158. https://doi.org/
10.1038/s41581-019-0110-2.
Van Bergen, T., Vandewalle, E., Van de Veire, S., Dewerchin, M., Stassen, J.M.,
Moons, L., Stalmans, I., 2011. The role of different VEGF isoforms in scar formation
after glaucoma filtration surgery. Exp. Eye Res. 93, 689–699. https://doi.org/
10.1016/j.exer.2011.08.016.
Wang, J., Li, W., Zhao, Y., Kang, D., Fu, W., Zheng, X., Pang, X., Du, G., 2018. Members
of FOX family could be drug targets of cancers. Pharmacol. Ther. 181, 183–196.

https://doi.org/10.1016/j.pharmthera.2017.08.003.

Yamanaka, O., Kitano-Izutani, A., Tomoyose, K., Reinach, P.S., 2015. Pathobiology of
wound healing after glaucoma filtration surgery. BMC Ophthalmol. 15 (Suppl. 1),
157. https://doi.org/10.1186/s12886-015-0134-8.
Yamanaka, O., Saika, S., Ikeda, K., Miyazaki, K., Ohnishi, Y., Ooshima, A., 2006.
Interleukin-7 modulates extracellular matrix production and TGF-beta signaling in
C. Lan et al.
Experimental Eye Research 210 (2021) 10872514
cultured human subconjunctival fibroblasts. Curr. Eye Res. 31, 491–499.
Yang, J., Feng, X., Zhou, Q., Cheng, W., Shang, C., Han, P., Lin, C., Chen, H.,
Quertermous, T., Chang, C., 2016. Pathological Ace2-to-Ace enzyme switch in the
stressed heart is transcriptionally controlled by the endothelial Brg1-FoxM1
complex. Proc. Natl. Acad. Sci. U. S. A. 113, E5628–E5635. https://doi.org/
10.1073/pnas.1525078113.
Zada, M., Pattamatta, U., White, A., 2018. Modulation of fibroblasts in conjunctival
wound healing. Ophthalmology 1251, 79–192. https://doi.org/10.1016/j.
ophtha.2017.08.028.
Zeng, R., Lu, X., Lin, J., Hu, J., Rong, Z., Xu, W., Liu, Z., Zeng, W., 2019. Knockdown of
FOXM1 attenuates inflammatory response in human osteoarthritis chondrocytes. Int.
Immunopharm. 68, 74–80. https://doi.org/10.1016/j.intimp.2018.12.057.
Zhang, F., Liu, K., Cao, M., Qu, J., Zhou, D., Pan, Z., Duan, X., Zhou, Y., 2019.
Rosiglitazone treatment prevents FDI-6 postoperative fibrosis in a rabbit model of
glaucoma filtration surgery. Invest. Ophthalmol. Vis. Sci. 60, 2743–2752. https://
doi.org/10.1167/iovs.18-26526.
Zhang, Y., Cheng, C., Wang, S., Xu, M., Zhang, D., Zeng, W., 2019. Knockdown of FOXM1
inhibits activation of keloid fibroblasts and extracellular matrix production via
inhibition of TGF-beta1/Smad pathway. Life Sci. 232, 116637.