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ORIGINAL RESEARCH
published: 04 June 2021
doi: 10.3389/fcvm.2021.649124
Frontiers in Cardiovascular Medicine | www.frontiersin.org 1June 2021 | Volume 8 | Article 649124
Edited by:
Masuko Ushio-Fukai,
Augusta University, United States
Reviewed by:
Ha Won Kim,
Augusta University, United States
Margarethe Geiger,
Medical University of Vienna, Austria
Sudhahar Varadarajan,
Augusta University, United States
*Correspondence:
Hua Zhu
hua.zhu@osumc.edu
Alexandra R. Lucas
arlucas5@asu.edu
†These authors have contributed
equally to this work
‡Present address:
Jordan R. Yaron,
School for Engineering of Matter,
Transport and Energy, Ira A. Fulton
Schools of Engineering, Arizona State
University, Tempe, AZ, United States
Specialty section:
This article was submitted to
Atherosclerosis and Vascular
Medicine,
a section of the journal
Frontiers in Cardiovascular Medicine
Received: 03 January 2021
Accepted: 28 April 2021
Published: 04 June 2021
Citation:
Ju B, Guo O, Benissan-Messan DZ,
Shawver MH, Chen P, Geng B, Wei S,
Yaron JR, Lucas AR and Zhu H (2021)
Serp-1 Promotes Corneal Wound
Healing by Facilitating
Re-epithelialization and Inhibiting
Fibrosis and Angiogenesis.
Front. Cardiovasc. Med. 8:649124.
doi: 10.3389/fcvm.2021.649124
Serp-1 Promotes Corneal Wound
Healing by Facilitating
Re-epithelialization and Inhibiting
Fibrosis and Angiogenesis
Brent Ju 1†, Owen Guo 1† , Dathe Z. Benissan-Messan 1, McKinley H. Shawver 1, Peng Chen 1,
Bingchuan Geng 1, Siqi Wei 1, Jordan R. Yaron 2‡, Alexandra R. Lucas 2
*and Hua Zhu 1
*
1Division of Cardiac Surgery, Department of Surgery, The Ohio State University Wexner Medical Center, Columbus, OH,
United States, 2Center for Personalized Diagnostics and Center for Immunotherapy, Vaccines and Virotherapy, Tempe, AZ,
United States
Purpose: Chemical corneal injuries carry a high morbidity and commonly lead to visual
impairment. Here, we investigate the role of Serp-1, a serine protease inhibitor, in corneal
wound healing.
Methods: An alkaline-induced corneal injury was induced in 14 mice. Following injury,
five mice received daily topical saline application while nine mice received Serp-1
100 µL topically combined with a daily subcutaneous injection of 100 ng/gram body
weight of Serp-1. Corneal damage was monitored daily through fluorescein staining and
imaging. Cross sectional corneal H&E staining were obtained. CD31 was used as marker
for neovascularization.
Results: Serp-1 facilitates corneal wound healing by reducing fibrosis and
neovascularization while mitigating inflammatory cell infiltration with no noticeable harm
related to its application.
Conclusions: Serp-1 effectively mitigates inflammation, decreases fibrosis, and reduce
neovascularization in a murine model of corneal injury without affecting other organs.
Translational Relavence: Our study provides preclinical data for topical application of
Serp-1 to treat corneal wounds.
Keywords: corneal injury, neovascularization, Serp-1, PAI-1, corneal wound repair
INTRODUCTION
Corneal injuries are a commonly cited reason for emergency department visits in the United States
(1,2). Although they are typically non-fatal, they can result in significant morbidity and change
in quality of life. Ocular chemical burn in particular, represent an ophthalmic emergency and
constitute about 11.5–22.1% of eye injuries (3). The most common causes of ocular chemical
burns are household cleaners and building products such as ammonia, sodium hydroxide, and
plaster (2). Accidents involving these chemicals require prompt diagnosis and frequently result in
hospitalization for treatment (1,2).
Ju et al. Serp-1 Promotes Corneal Healing
Under normal circumstances, the corneal epithelium acts as a
protective barrier to the ocular bulb (2). Intercellular attachments
and attachments of epithelial cells to the extracellular matrix
through both junctional and non-junctional adhesions, maintain
this barrier (4). With damage, epithelial cells undergo apoptosis
and are shed in tear films (2). Intercellular junctions are
disrupted, and cell-substrate junctions are temporarily replaced
with weaker attachments and a provisional extracellular matrix is
laid down in preparation for repair (5). Chemical ocular injuries
are particularly more deleterious due to the additional oxidative
stress they impose on the cornea which not only damages
the corneal cells but will also trigger an immune response
characterized by inflammation (2,6). This damage to the cornea
causes a keratocyte induced fibrosis which hardens and opacifies
the cornea and results in varying degrees of blindness (7).
Additionally, while acute inflammation is initially beneficial to
the eye, long-term sequalae of inflammation in the cornea include
neovascularization which can threaten vision (8).
Serp-1 is a myxoma virus derived SERine Protease INhibitor
(SERPIN) with previously demonstrated roles in inflammation,
cell migration, wound closure, tissue remodeling and fibrosis (9–
12). It is a single-chain glycosylated protein composed of three
β-sheets and nine α-helical domains with a strained reactive
center loop (RCL) positioned in the carboxy terminus (13).
SERPINs are important injury response factors that participate
in all stages of injury repair [summarized in our recent review
article (14)]. We have previously showed that recombinant Serp-
1 protein could be a potential therapeutic agent to reduce
aortic balloon angioplasty injury (15), suppress atherosclerotic
plaque growth (16), prevent chronic renal (17), and cardiac (18)
allograft rejection, promote spinal cord injury repair (19) and full
thickness dermal wound healing (10) in rodent models. Here, we
investigate the role of Serp-1 in corneal wound healing through
immune system modulation.
METHODS
Animal Corneal Wound Healing Model
Animal husbandry and experimentations were conducted with
approval from the Institutional Animal Care and Use Committee
(IACUC) at The Ohio State University. All injuries were induced
under anesthesia (Henry Schein Isothesia, Isofluorane). Four
milliliters of Ibuprofen (Perrigo Basic Care, 100 mg per 5 mL
Oral Suspension) was mixed into 400 mL ddH2O (Millipore
Sigma Milli-Q IQ system) and made accessible to the mice
24 h before the injury and throughout the duration of the
entire experiment. A solution of buprenorphine was made by
triturating buprenorphine and saline solution (Baxter, 0.9%
Sodium Chloride) in a 3:50 ratio by volume. Each mouse received
a 100 µL subcutaneous injection of the buprenorphine solution
every 12 h following the injury for 72 h.
Fourteen mice (C57BL/6J) were selected for this experiment.
A piece of filter paper, 2-mm in diameter, was soaked in
1M NaOH solution and placed on the cornea of the mouse’s
right eye for 30 s and then rinsed off with 15 mL of saline
solution (Baxter, 0.9% Sodium Chloride). The clinical opacity
and neovascularization scores were determined using Modified
Hackett-McDonald scoring methods for 10 days after the injury
(20). After the analysis was complete, the mice were sacrificed,
and the eyes were collected for flat-mount and paraffin-
embedded staining. The size and depth of the corneal damage
was monitored and recorded daily through fluorescein staining
and imaging. Ten microliters of fluorescein was placed on the
right eye of the mouse and the excess fluorescein was rinsed off
with saline. The Kowa SL-17 slit lamp was set to the cobalt blue
filter and used to illuminate the eye and excite the fluorescein
particles so that they could be captured by a point and shoot
camera (Samsung ST150F). Images were obtained once per day.
For treatment of the corneal wounds, the mice were divided
into two experimental treatment groups. One group received a
control treatment of 10 µL of phosphate-buffered saline (PBS)
applied topically for 10 min. The second group not only received
a 10 µL (0.1 µg/µl) topical treatment of recombinant Serp-1
protein but also a subcutaneous injection of recombinant Serp-
1 protein (0.1 µg/µl) in a dosage equivalent to 100 ng per gram
of body weight (recorded every morning) based on our previous
publication (10). Treatments were applied twice daily for a total
of 10 days following the injury. Mice body weights and eating
habits were monitored daily and used as indicators of health
status. Heart, lung, kidney, liver, splenic tissues were collected at
the completion of the experiment and evaluated for pathology.
Histopathology and Immunohistochemistry
The mice were sacrificed following 10 days of observations.
The eyes were removed, set in paraformaldehyde (BioWorld,
4% in PBS, pH 7.4) overnight, and then transferred into 70%
ethanol. The corneas were dissected from the ocular bulb and
placed into a 96-well plate with 100 µL PBS (Thermo Fisher
Scientific). Corneas were washed 3 times with 300 µL PBS for
10 min per wash and placed in blocking buffer solution for 2h at
room temperature. Anti-CD31 antibodies (1:100, BD Biosciences
Pharmingen) was applied, followed by overnight incubation at
4◦C. All corneas were washed 6 times with 1x washing buffer
for 1 h at a time at room temperature. Secondary antibodies
α-rat 488 (1:500, 1:500, Thermo Fisher Scientific) was applied
for CD31(angiogenesis/neovascularization marker. The corneas
were then washed 3 times with PBS for 1 h each. Four cuts were
made on the cornea to “butterfly” the sample so that the sample
could be laid down in a flat manner. Mounting was performed
in mounting buffer containing 4′,6′-diamidino-2-phenylindole
(DAPI). Additionally, full corneal cross-sections were obtained,
embedded in paraffin, and stained using Hematoxylin and Eosin
(H&E). Immunofluorescent staining of CD11b [anti-CD11b
antibody (Invitrogen, 14-0112-82, 1:200)] was performed as
follows: slides were deparaffinized and rehydrated by incubating
sequentially in xylene, 100% ethanol, 95, 75, 50% ethanol and
PBS. A pressure cooker was used for antigen retrieval. Slides were
merged in Tris-EDTA buffer and cooked for 13 min. Primary
antibody were applied and incubated at 4◦C overnight. Goat anti-
rat secondary antibody Alexa-488 (Invitrogen, A11006) were
applied and incubated at room temperature for 1 h. All images
were captured by a Zeiss LSM 780 confocal microscope and
analyzed by ImageJ as described in our previous publication (21).
Frontiers in Cardiovascular Medicine | www.frontiersin.org 2June 2021 | Volume 8 | Article 649124
Ju et al. Serp-1 Promotes Corneal Healing
Opacity and Neovascularization Scores
A modified Hackett-McDonald scoring was used (20). Image J
was used for immunofluorescence image analysis. Vessel density
was calculated by tracing the fluorescent, CD31 positive areas in
ImageJ then dividing that area by the total cornea area to achieve
a percentage for vessel coverage.
Production of Recombinant Serp-1 Protein
Recombinant Serp-1 protein was produced by Chinese hamster
ovary (CHO) cell line protein expression system (Viron
Therapeutics Inc., London, ON, CA), as described by our
previous publication (10). Sequential column chromatographic
separation was employed to purify GMP-compliant recombinant
Serp-1 protein. Purity of Serp-1 protein (>95%) was determined
by Coomassie-stained SDS-PAGE and reverse-phase HPLC.
Endotoxin was confirmed absent from purified Serp-1 by LAL
assay (10).
Statistical Analysis
The data are represented as mean ±standard deviation.
Comparisons were made by Student’s t-test when comparing
two experimental groups. The standard deviation of the mean is
indicated by error bars for each group of data. A value of p<0.05
was considered significant. All of these data were analyzed using
Prism 8 software.
RESULTS
Serp-1 Facilitates Corneal Wound Healing
by Reducing Fibrosis and
Neovascularization
To test the effectiveness of Serp-1 as a treatment for corneal
injuries, an alkaline-induced corneal injury was induced in 14
mice. Following injury, a control group of five mice received
daily topical saline application while nine mice received Serp-1
(100 ng/ µL concentration, 100 µL topically plus 100 ng/gram
body weight subcutaneously) each day. Fluorescein dye and
quantitative opacity and neovascularization scores were used
to determine the level of fibrosis, neovascularization, and re-
epithelialization in the corneas.
Fluorescein positive-areas and fluorescence intensity were
used as indicators for corneal damage and re-epithelialization.
Mice who received Serp-1 treatment exhibited smaller
fluorescein-positive areas and lower intensity fluorescence when
compared to the mice who received only saline (Figure 1A).
A significant difference between the damaged areas was noted
by Day 5 following injury (p<0.05) (Figure 1B). Serp-1
treated mice corneas consistently showed reduced opacity
and vascularization, with a significant difference in both
opacity and neovascularization starting from Day 6 post-injury
(Figures 1C,D,∗:P<0.05; ∗∗:P<0.01). CD31 expression
was used as an indicator of the presence of superficial and deep
neovascularization in corneal sections. Evaluation of the CD31
staining revealed a reduction in neovascularization in the Serp-1
treated group (Figure 2,∗:P<0.05).
Serp-1 Reduces Rates of Inflammatory
Cell Infiltration Into the Cornea
Histochemical analysis of the enucleated globes of both Serp-
1 and control-treated mice was performed following 10 days
of observations. Cross-sectional image analysis following H&E
staining revealed an obvious reduction of corneal swelling
and decreased inflammatory cells infiltration in Serp-1 treated
animals (White arrows in Figure 3A). Similarly, CD11b positive
immune cells were stained. We found that Serp-1 treatment
significantly reduced immune cell infiltration following alkali
burn injury (Figures 3B,C,∗:P<0.05).
Repetitive Application of Serp-1 Produces
No Obviously Toxic Effects
Since Serp-1 protein is a virus derived protein, we also checked
whether repetitive administration of Serp-1 could induce any
adverse effects to the mice. We first checked mouse body weight
change after Serp-1 injection. While the mice receiving saline
gained body weight, we did observe Serp-1 treatment group had
slight reduction of body weight (Figure 4A). However, when
we checked major organs of the mice treated with Serp-1, we
didn’t observe pathologic inflammatory changes as result of the
use of Serp-1 (Figure 4B). Thus, further studies will be required
to identify the cause of slight body weight loss after repetitive
administration of Serp-1 protein in mice.
DISCUSSIONS
The application of Serp-1 following corneal alkali induced
injury leads to reduced fibrosis and neovascularization, and a
decrease in inflammatory cell infiltration. In order to maintain
transparency, the cornea must remain avascular and unscarred
(22). Since chemically induced corneal injuries rapidly progress
to visual impairment, attention has recently turned toward
developing treatments for rapid re-epithelialization of the cornea
with minimal fibrosis and neovascularization. The novelty of this
study is evident in the fact that it demonstrates the therapeutic
efficacy of Serp-1 in promoting the natural healing process of the
cornea following injury in a mouse model of corneal injury.
We hypothesized that Serp-1 facilitates corneal wound healing
by reducing inflammation. An important facet of corneal wound
healing is the rate at which the cornea re-epithelialize (22). To
monitor re-epithelialization in the mice, fluorescein dye was used
daily to stain areas of the mouse cornea where the tight cell-
to-cell junctions of the epithelium were compromised. Analysis
during a period of 10 days following injury revealed quick healing
in mice treated with Serp-1. These results are consistent with
the findings from previous work done in evaluating the role
of Serp-1 in dermal wound healing (10). The reduced rates of
opacity and neovascularization in the Serp-1 treatment group
demonstrate a reduced inflammatory response. Additionally,
observations from our paraffin-embedded cross sections revealed
reduced corneal swelling and improved morphology with Serp-1
treatment following injury which points to an improved rate of
corneal epithelialization.
Frontiers in Cardiovascular Medicine | www.frontiersin.org 3June 2021 | Volume 8 | Article 649124
Ju et al. Serp-1 Promotes Corneal Healing
FIGURE 1 | Serp-1 contributes to the corneal wound healing process by limiting the inflammatory response. (A) Treatment with Serp-1 shows improved
re-epithelialization in mouse corneas with an alkaline induced injury (n=5 for control group and n=9 for Serp-1 group). (B) Quantification of fluorescent signal in (A)
by dividing the fluorescein positive area by total corneal area (data were presented as mean ±S.D. *P<0.05). (C) Bright-field imaging shows reduced fibrosis and
encroachment of the cornea by neovascularization in mice treated with Serp-1. (D) Quantification of corneal fibrosis and neovascularization using a modified
Hackett-McDonald scoring system (data were presented as mean ±S.D. *P<0.05; **P<0.01).
FIGURE 2 | Immunostaining with flat-mounted corneas shows reduced inflammation in Serp-1 treated mice. Immunostaining with flat-mounted corneas shows
reduced inflammation in Serp-1 treated mice. Anti-CD31 antibody was applied and samples were mounted in buffer containing 4′,6′-diamidino-2-phenylindole (DAPI).
Total vessel area was quantified by dividing the CD31 positive areas by the total cornea area using FijiWin’s ImageJ software, with a significant difference reported
between the two treatment groups (n=3/group; *p<0.05).
In addition to a fast recovery time, it is essential for the
cornea to heal without excessive fibrosis or neovascularization
which impede light transmission. Due to its anti-inflammatory
properties, it was hypothesized that injured corneas treated
with Serp-1 would have reduced amounts of fibrosis and
neovascularization. Our data points toward a significant
reduction in opacity and neovascularization in animals treated
with Serp-1. This was further confirmed through quantitative
analysis of the total area of neovascularization in flat-mount
staining. Together, these data support the hypothesis that Serp-1
inhibit the inflammatory response in a positive manner that
limits fibrosis and scarring that impairs vision.
The immunogenicity of Serp-1 is a legitimate concern due
to the fact that it is virally derived. Daily monitoring of mice’s
body weights and staining of other major organ tissue in
these experiments revealed no cytotoxic impact of the repeated
application and injection of Serp-1. This finding is entirely
consistent with prior research both in pre-clinical or clinical
studies wherein Serp-1 demonstrated no significant toxicity
and no neutralizing antibodies were detected in the Phase
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Ju et al. Serp-1 Promotes Corneal Healing
FIGURE 3 | Histochemical analysis of eye cross-sections show reduced rates of inflammatory cell infiltration and swelling of the cornea in Serp-1 treated mice. (A)
Histochemical analysis of eye cross-sections shows reduced rates of inflammatory cell infiltration and swelling of the cornea in Serp-1 treated mice. Enlarged images
of the corneal region of two mice visibly show more swelling and a higher presence of inflammatory cells in the control mice. Inflammatory cell nuclei are stained by the
deep-purple spots marked with white triangles in the light pink stromal layer of the cornea. (B) To further identify infiltrating immune cells, the slides were stained with
CD11b (an immune cell marker, green). (C) CD11b positive cells (cells/0.1 mm2) were quantified (n=3/group; *p<0.05).
FIGURE 4 | Repetitive application of Serp-1 produces no obviously toxic effects. (A) Changes of body weight before and after experiments were measured and
calculated (n=5 for control group, n=9 for Serp-1 group. *P<0.05). (B) Histochemical analysis of eye cross-sections shows reduced rates of inflammatory cell
infiltration and swelling of the cornea in Serp-1 treated mice. Enlarged images of the corneal region of two mice visibly show more swelling and a higher presence of
inflammatory cells in the control mice. Inflammatory cell nuclei are stained by the deep-purple spots marked with white triangles in the light pink stromal layer of the
cornea.
2 clinical trial in patients with coronary stent implant (23).
Future directions as a result of this analysis should include
evaluation in larger samples, evaluation of topical applications
alone, analysis of interactions with the uPA, MMP proteases
after corneal injury and serp-1 treatment, evaluation in other
primates, and consideration for clinical trials in humans for an
improved understanding of the safety and efficacy of the protein.
Further studies should also aim to investigate the molecular
mechanism of action of Serp-1 in its anti-inflammatory role.
Finally, a recent publication from our group demonstrated that
administration of recombinant M-T7 protein (another Myxoma
virus protein) could also accelerate dermal wound healing (24).
This provides an additional example of a Myxoma virus derived
protein in facilitating tissue repair. Thus, future investigations are
needed to explore potential Myxoma virus proteins with tissue
repair properties.
Frontiers in Cardiovascular Medicine | www.frontiersin.org 5June 2021 | Volume 8 | Article 649124
Ju et al. Serp-1 Promotes Corneal Healing
There is a similar study by Liu et al. elegantly showed
topical application of serpinA3K could promote alkali induced
corneal wound healing via inhibiting neovascularization and
inflammation (25). Thus, it would be interesting to compare the
beneficial effects between mammalian serpin and viral serpin in
corneal wound healing.
While our study showed beneficial effects of administration
of Serp-1 to treat corneal wound in mice, there are some
limitations that we will address in our future studies. First,
our control treatment group did not use an inactive mutant
protein, thus, the beneficial effect we observed in Serp-1
group might be simply due to general protein effect. Our
previous publication identified an inactive Serp-1 mutant
(26), which would be a preferred negative control for our
future study. We are currently working on development
of purification protocol for the GMP level mutant Serp-1
for our in vivo animal studies in the future. Second, one
of direction of our future studies will focus on dissecting
the weight loss observe in Serp-1 treatment group. For
example, different doses of Serp-1 should be tested and
different administration methods should be compared (topical
application, intravenous injection, intraperitoneal injection and
subcutaneous injection, etc.). Finally, comprehensive toxicology
analysis should be included, such as inflammatory cytokine
analysis, liver function analysis, potential cardiovascular function
analysis and systemic/metabolic disruption of adipose tissue
functions or circulating lipids level should be measured.
CONCLUSIONS
Serp-1 can modulates and enhance the corneal wound healing
response. The protein can effectively mitigate inflammation,
decrease fibrosis, and reduce neovascularization in the cornea
following alkaline-induced injuries in a murine model. Further
studies to investigate the biologic mechanism of action of Serp-1
are needed to further outline its role, its safety profile, and define
its potential as an alternative to standard of care in the treatment
of corneal injuries.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation.
ETHICS STATEMENT
The animal study was reviewed and approved by IACUC of the
Ohio State University.
AUTHOR CONTRIBUTIONS
BJ, OG, MS, BG, SW, and HZ performed the experiments. ARL
and HZ designed the studies. BJ, OG, and HZ analyzed the data.
BJ, OG, DB-M, JRY, ARL, and HZ wrote the paper. All authors
contributed to the article and approved the submitted version.
FUNDING
This work was partially supported by a grant from National
Eye Institute to HZ (EY030621), from the American Heart
association (AHA) 17GRNT33460327 to ARL, National Institutes
of Health (NIH) R01AR074627-01A1 to ARL, and a post-
doctoral Ruth L. Kirschstein National Research Service Award to
DB-M (2T32AI106704-06).
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Copyright © 2021 Ju, Guo, Benissan-Messan, Shawver, Chen, Geng, Wei, Yaron,
Lucas and Zhu. This is an open-access article distributed under the terms of
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