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Attenuation of corneal
neovascularization by topical low-molecular-weight heparin-taurocholate 7
without bleeding complication
Jae Yong Kim1, Soo Yeon
Kim1, Mi Hyun Cheon1, Eun-Soon Kim1, In Seok
Song2, Myoung Joon Kim1, Hungwon Tchah1
1Department of Ophthalmology,
University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea
2Department of Ophthalmology, Hanyang
University College of Medicine, Seoul 04763, Korea
Co-first authors: Jae Yong Kim and Soo Yeon Kim
Correspondence to: Hungwon
Tchah.
Department of Ophthalmology, University of Ulsan College of Medicine, Asan
Medical Center, 388-1 Pungnab-2dong, Songpa-gu, Seoul 05505, Korea. hwtchah@amc.seoul.kr
Received: 2015-01-01
Accepted: 2015-12-08
Abstract
AIM: To investigate the antiangiogenic
effects and safety of topically administered low-molecular-weight
heparin-taurocholate 7 (LHT7) on corneal neovascularization (CoNV).
METHODS: Twenty-four Sprague-Dawley rats were randomly distributed into four groups of six rats
each. The central corneas were cauterized using a silver/potassium nitrate
solution. From 2d after cauterization, 12.5 mg/mL (low LHT7 group) or 25 mg/mL (high LHT7 group) LHT7 was
topically administered three times daily; 12.5 mg/mL bevacizumab was topically
administered as positive control (bevacizumab) group, with normal saline (NS)
administered as negative control (NS group). The
corneas were digitally photographed to calculate the CoNV percentage from the
neovascularized corneal area at 1 and 2wk.
RESULTS: The 4 study groups did not
have different CoNV percentages at 1wk after injury (P>0.05). However, the low LHT, high LHT, and bevacizumab groups had
significantly lower CoNV percentages than the NS group at 2wk (all P<0.05). No significant differences in CoNV
percentage were found among the low LHT, high LHT, and bevacizumab groups (all P>0.05). All groups except the NS group had lower CoNV percentages at 2wk
post-injury than the levels observed at 1wk (all P<0.05).
CONCLUSION: Topically-administered
LHT7 inhibited CoNV without complication after chemical cauterization in the
rat.
KEYWORDS: bevacizumab; chemical cauterization; corneal neovascularization; low-molecular-weight heparin-taurocholate 7
DOI:10.18240/ijo.2016.09.03
Citation: Kim
JY, Kim SY, Cheon MH, Kim ES, Song IS, Kim MJ, Tchah H. Attenuation of
corneal neovascularization by topical low-molecular-weight heparin-taurocholate
7 without bleeding complication. Int J Ophthalmol 2016;9(9):1255-1259
INTRODUCTION
In order to control corneal neovascularization
(CoNV), various strategies including the medical modalities including the
application of steroids,
cyclosporin A, methotrexate, and non-steroidal anti-inflammatory drugs;
photodynamic therapy; fine needle diathermy; argon laser photocoagulation and
have been attempted[1-9]. The systemic or intravitreal
anti-vascular endothelial growth factor (VEGF) monoclonal antibody,
bevacizumab (Avastin; Roche, Basel, Switzerland) is injected to successfully
treat retinopathy of prematurity, age-related macular degeneration, and macular
edema caused by diabetic retinopathy and retinal vein occlusion[13-16].
The topical administration of bevacizumab can inhibit CoNV[10-12].
Heparin antagonizes blood clotting by creating
a complex involving antithrombin III[17-18]. Heparin sulfate binding to growth factors stabilizes them and
attenuates their activation by blocking their diffusion and diminishing
proteolytic degradation[19-20]. Heparin derivatives are created to not only attenuate hemorrhagic
adverse effects but also strengthen the beneficial effects of heparin, such as
anti-angiogenesis. Low-molecular-weight heparin-taurocholate 7 (LHT7) is an low-molecular weight
heparin (LMWH) conjugated with seven
taurocholates, having a polyproline helical structure with more negative
charges and facilitating stronger binding to growth factors, including VEGF,
than other heparin derivatives[21]. The LHT7 molecule shows unique
features that strongly attenuate VEGF165-dependent angiogenesis by
antagonizing phosphorylation of the VEGF165 receptor. However,
bevacizumab is a monoclonal antibody directly against VEGF165. We
recently showed that the subconjunctival LHT7 administration attenuates CoNVs
using rat chemical cauterization, despite complications that included corneal
stromal hemorrhage[22]. This suggested that topical
applications of LHT7 should be considered to overcome this adverse effect. In
our present study, we attempted to investigate the effects and safety of
topically administered LHT7 on CoNV using the same rat chemical cauterization.
So far, the antiangiogenic effects and safety of topical LHT7 have not
previously been determined.
MATERIALS AND METHODS
All animal were managed in accordance with the
guidelines of the Association for Research in Vision and Ophthalmology
Statement for the Use of Animals in Ophthalmic and Vision Research. The
experimental protocol was approved by the Institutional Animal Care and Use
Committee of Asan Medical Center, Seoul, Korea.
Twenty eight healthy 5- to 6-week-old Sprague-Dawley
rats weighing 225 g to 275 g were included. Under the deep anesthesia induced
by intraperitoneal xylazine hydrochloride (10 mg/kg) administration, the whole
procedures were carried out. In addition with topical anesthesia induced by
0.5% (wt/vol) proparacaine hydrochloride (Alcaine; Alcon laboratories, Fort
Worth, TX, USA) administration, a chemical injury was created 3 mm in diameter
by touching an 75% (wt/vol) silver nitrate/25% (wt/vol) potassium nitrate
(Arzol Chemical, Keen, NH, USA)-coated applicator stick onto the central cornea
for 8s[23]. Vigorous irrigation was carried out with 10 mL of
balanced salt solution (Alcon Laboratories) to remove remained silver
nitrate/potassium nitrate solution. All chemical injuries were created by a
single researcher to maintain the consistency of chemical injury.
Fourty-eight hours after the injury, the corneal burn injuries were scored as
previously reported[20]. Only corneas with burn injury
scores of equal or greater than +2 were involved in the evaluation of CoNV
score[24]. Four of 28 eyes were excluded in the first week
after the injury due to immoderate intraperitoneal anesthesia. Thus, 24 rats
were randomly divided to one of four groups after cauterization: the low LHT7
group (n=6) received 0.02 mL of 12.5
mg/mL of LHT7 topically and the high LHT7 group (n=6) received 0.02 mL of 25 mg/mL of LHT7 topically. With regard to
randomization, mice were randomized using a stratified design according to
which cage they lived. Within each cage, they were randomly assigned to one of
four groups. As a positive control, rats in the bevacizumab group (n=6) received 0.02 mL of 12.5 mg/mL of
bevacizumab topically, whereas those in the normal saline (NS) group (n=6) received 0.02 mL of 0.9% (wt/vol))
of NS topically as a negative control. In all groups, treatment eyedrops were
topically administered three times a day for 14d and started at 48h after
injury induction in all groups. The bevacizumab concentration was chosen based
on previous reports to facilitate inter-study comparisons[12,25]. Topical
administrations were performed using 20 µg micropipettes (Pipetman P;
Gilson, Inc., Paris, France). All LHT7 and bevacizumab eyedrops were made with
sterile NS. LHT7 was kindly given by Professor Youngro Byun, College of
Pharmacy, Seoul National University, Seoul, Korea.
All eyes were checked using an optical
microscopy at 1 and 2wk after injury under the deep anesthesia. All corneas
were digitally photographed using a digital camera (32¡¿ magnification; Coolpix 4500, Nikon Imaging Japan,
Tokyo, Japan). The image analysis for each cornea was carried out using the
image software program (Image J; v.1.40; National Institute of Mental Health,
Bethesda, MD, USA). We calculated the area of the CoNV in pixels and scored the
proportion of this neovascularized area with respect to the whole cornea as the
CoNV percentage[5,26-29]. After these calculations
for all groups, the animals were sacrificed on week 2.
Data
were shown as the means¡¾standard errors (SE). Statistical
analyses were carried out to compare CoNV percentages and changes in the CoNV
among four groups with the paired Wilcoxon-signed rank test, the Mann-Whitney U
test, and analysis of variance (ANOVA) using the SPSS statistics program
(version 13.0; SPSS, Chicago, IL, USA). The 95% confidence was obtained as P<0.05.
RESULTS
Two days after cauterization, the four study
groups did not have different burn injury scores (P=0.83).
The CoNV percentage levels at 1 and 2wk after chemical injury (Table 1, Figure 1). All four groups did
not have significantly different percentages of CoNV at 1wk post-injury (P>0.05). However, the low LHT, high LHT and bevacizumab groups had significantly lower CoNV
levels at 2wk post-injury
than the
NS control group (all P<0.05). No significant differences in CoNV
percentage were found among the low LHT, high LHT, and bevacizumab groups (all P>0.05) (Table 1; Figure 1A). All groups except the NS
group had significantly lower CoNV percentages at 2wk than the levels observed at 1wk post-injury (all P<0.05; Table
1; Figure 1B). No
adverse effects such as corneal perforation or corneal stromal hemorrhage were
observed in any group after cauterization (Figure 2).
Table 1 The low LHT, high LHT and
bevacizumab groups had significantly lower CoNV percentages than the NS group
Percentage of CoNV, % |
Low LHT7 group |
High LHT7 group |
Bevacizumab group |
NS group |
1wk 2wk |
19.2¡¾4.8 6.2¡¾2.5a |
31.8¡¾13.2 7.3¡¾1.6a |
53.9¡¾11.9 16.5¡¾7.8a |
61.2¡¾14.3 55.9¡¾11.8a |
Difference between 1 and 2wk |
-13.0¡¾2.5b |
-24.5¡¾12.7b |
-37.4¡¾10.9b |
-0.5¡¾4.9 |
aSignificant difference
among four groups (P<0.05); bSignificant
difference in CoNV between 1 and 2wk (P<0.05).
Figure
1 The low LHT, high LHT and
bevacizumab groups had significantly lower CoNV percentages than the NS group aSignificant difference among
four groups (P<0.05); bSignificant difference
in CoNV between 1 and 2wk (P<0.05).
Figure 2 Slit lamp microscopic photographs Slit
lamp microscopic photographs showing CoNV in eyes treated with LHT7 (low and
high LHT7 groups), bevacizumab-treated eyes (bevacizumab group), and normal
saline-treated control eyes (NS Group) at 1wk (A, B, C and D) and 2wk (E, F, G and H) after
chemical cauterization.
DISCUSSION
Topical bevacizumab administration has been
performed previously for the treatment of CoNV in rats[24,30-32] and
rabbits[33-34]. In our current study, the bevacizumab group,
receiving 12.5 mg/mL of this drug topically, was included as a positive
control, whilst the NS group as a negative control. The same concentration of
topical LHT7 as topical bevacizumab was determined to use in low LHT group,
because our
previous study showed that the anti-angiogenicity of subconjunctivally
administed LHT7 is equivalent to that of subconjunctivally administered
bevacizumab[1]. In high LHT group, 25 mg/mL topical LHT was used to
observe dose-dependency. Our results suggest that the
antiangiogenic effects of a topical application of LHT7 are marginally superior
to those of bevacizumab, although this difference did not reach statistical
significance (Table 1; Figure 1).
Comparing our current results with those
reported previously for subconjunctival LHT7 injection[22], there was a
significant difference found in the percentage of CoNV at 2wk after
cauterization following topical LHT7 administration. This suggested that for
LHT7, a topical treatment produces a more stable and consistent antiangiogenic
effect than a subconjunctival injection. Topically administered bevacizumab has
been reported to show a longer lasting antiangiogenic effect than
subconjunctivally injected bevacizumab in CoNV after chemical injury in rats[12].
However, Hashemian et al[31] have reported that both subconjunctival and
topical bevacizumab have equal potency in preventing CoNV in rats.
In terms of complications in our current study,
we observed no adverse effects, such as corneal perforation or corneal stromal
hemorrhage, in our LHT groups. Conversely, we have previously reported two
cases of corneal stromal bleeding, one in each of a low and high LHT7 group
that subconjunctivally received 0.02 and 0.04 mL of 25 mg/mL of LHT7,
respectively[22]. Hence, the topical administration of LHT7
is proving to be less invasive and safer than the subconjunctival injection of
this compound.
Our study had several limitations of note.
First, the followed-up period was only 2wk after chemical cauterization, which
was a relatively short. Second, we did not determine the optimal dosage for
treating CoNV by topical LHT7 and further studies are needed to evaluate the
critical range of LHT7 concentrations to use in a clinical application.
Finally, the corneas should have been immunohistochemically stained with
special antibodies including anti-CD31 antibody after whole mounts to get more
accurate results[35], despite CoNVs can be clearly
investigated in the rat chemical cauterization model. We conclude from our
current analyses, however, that the topical application of LHT7 efficiently attenuates CoNV after
chemical cauterization in the rat without producing adverse effects.
ACKNOWLEDGEMENTS
Foundations: Supported by the Student Research Grant of University of Ulsan College
of Medicine, Seoul, Korea (No. 12-13); the Asan Institute for
Life Sciences, Seoul, Korea (No. 2014-464).
Conflicts of Interest: Kim JY,
None; Kim SY, None; Cheon MH, None; Kim ES, None; Song IS,
None; Kim MJ,
None; Tchah H, None.
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