infectious and inflammatory processes , however , can induce new vessel growth causing corneal neovascularization , which leads to scarring , edema , and blindness .
corneal neovascularization affects an estimated 4.1% of all patients presenting to general ophthalmology offices in the us
a meta - analysis has shown a significantly higher risk of corneal graft rejection with increased number of corneal quadrants affected by neovascularization prior to keratoplasty .
corneal neovascularization is thought to occur through the imbalance of angiogenic and antiangiogenic protein factors .
vascularized human corneas have been shown to have significant upregulation of vascular endothelial growth factor ( vegf ) , matrix metalloproteinases ( mmp ) , and basic fibroblastic growth factor ( bfgf ) .
, san francisco , ca ) is a monoclonal antibody that targets human vegf ( vegf ) and has been studied as a potentially potent therapy for corneal neovascularization .
antiangiogenic monotherapy with a drug such as bv , which targets vegf alone , however , may not provide the best long - term therapy since inhibition of one antiangiogenic factor can result in the upregulation of others leading to acquired drug resistance .
in addition , lymphangiogenesis has been identified as an important process involved in the pathogenesis of corneal graft rejection .
endostatin , in contrast to bevacizumab , has been identified as a potent inhibitor of both neovascularization and lymphangiogenesis [ 68 ] .
another potential deficiency of bv therapy is its association with systemic side effects seen during trials of its use in age - related macular degeneration , including bleeding , hypertension , and stroke [ 912 ] .
thus , an angiogenesis inhibitor that blocks multiple promoters of angiogenesis with few systemic side effects is desired .
as a 20  kda fragment of collagen xviii , it modifies 12% of the human genome in order to downregulate angiogenesis with few systemic side effects [ 1315 ] . by affecting multiple angiogenic pathways simultaneously , endostatin may allow for a much lower possibility of drug resistance .
in contrast to the noted hypertension found with bv use in human studies [ 17 , 18 ] , endostatin was noted to decrease systolic blood pressure by almost 10  mmhg when dosed daily in animal studies .
endostatin was initially studied in preclinical models of corneal neovascularization and tumor models [ 20 , 21 ] . despite high expectations ,
human endostatin had a limited response in phase i and ii human cancer clinical trials largely due to two deficiencies .
second , approximately 50% of the recombinant human endostatin used in the clinical trials lacked four amino acids at the nh2 terminus , which resulted in a nonfunctional molecule due to an inability to bind zinc . in order to engineer a more stable form of endostatin
, fc - endostatin ( fce ) was developed by fusing endostatin to the fc region of an igg molecule .
the presence of the fc portion increases the half - life to greater than one week and conserves the molecule 's zinc affinity .
this increase in half - life is similar to the role of the fc domain in bv , which increases the molecule 's half - life to weeks rather than hours .
fce was recently shown to be effective in a rodent model of high - grade glioma using various delivery methodologies .
we set out to evaluate the safety and antiangiogenic efficacy of subconjunctival injection of fce using a rabbit vegf - induced corneal neovascularization model .
this paper is the first published assessment of fce as a potential therapy for corneal neovascularization .
eight male new zealand white rabbits , each weighing 4.5 to 6.5  kg ( robinson industry farms , mocksville , nc ) were used . care and treatment of all rabbits were in strict agreement with the association for research in vision and ophthalmology ( arvo ) statement for the use of animals in ophthalmic and vision research and with the approval of the johns hopkins university animal care and use committee .
rabbits were housed in standard animal facilities , one animal per cage , and given free access to food and baltimore city water . for intracorneal implantations and subsequent operative microscope examinations
the animals were anesthetized with a mixture of xylazine 10  mg / kg ( butler schein , dublin , oh ) and ketamine 2  mg / kg ( butler schein , dublin , oh ) .
the ocular surface was anesthetized with topical 0.5% proparacaine hydrochloride ( alcaine , alcon , fort worth , tx ) . pain control was provided , as needed , with subcutaneous injection of buprenorphine hcl 0.1  mg / kg ( bedford labs , bedford , oh ) .
ethylene - vinyl acetate copolymer ( 40% vinyl acetate by weight , elvax 40p ) ( du pont co. , wilmington , de ) was prepared as previously described .
briefly , human vegf ( peprotech , rocky hill , nj ) was incorporated into the ethylene - vinyl acetate copolymer ( evac ) matrix .
evac ( 130  mg ) was dissolved in methylene chloride ( 1.8  ml ) and vegf ( 20  g ) was added .
the mixture was poured into cylindrical glass molds measuring 5  mm  220  mm and placed in 20c for 48 hours and the methylene chloride was allowed to passively evaporate .
the vegf polymer was then cut into uniform pellets of size 1  mm  1  mm  0.5  mm .
rabbits were placed under general anesthesia and topical 0.5% proparacaine hydrochloride was applied to the corneal surface .
5% povidone - iodine was applied on the ocular surface for antisepsis . at the center of the cornea , a 2  mm horizontal incision was placed to the midstromal level using the bevel of a 16 gauge needle ( bd , franklin lakes , nj ) .
two micropockets were made from the incision towards 6 and 12 o'clock by dissecting the corneal stroma with an iris spatula ( figure 1 ) .
one vegf polymer was placed at the end of each micropocket 2  mm apart from the limbus .
a total of 32 vegf polymer pellets were placed ( 2 pellets per cornea , one at 6 o'clock and one at 12 o'clock ) .
each eye received a subconjunctival injection , one half of each dose was injected at 6 and 12 o'clock in proximity to each vegf polymer , with either 0.125  ml of 0.9% normal saline ( ns ) , 0.1  ml of 25  mg / ml bv , 0.125  ml of 20  mg / ml human fce ( fce 20 ) ( bio x cell , west lebanon , new hampshire ) , or 0.125  ml of 2  mg / ml human fce ( fce 2 ) .
a volume of 0.125  ml of fce was used in order to approximate the mg concentration of bv 25  mg / ml .
subconjunctival injections were performed twice weekly starting on the day of surgery : days 0 , 5 , 8 , and 12 .
corneal neovascularization was assessed via operative microscopy ( carl zeiss , germany ) on days 0 , 5 , 8 , 12 , and 15 .
the corneas were digitally imaged , and the images were analyzed using imagej software ( nih.gov ) . to have a single standardized value that incorporated both vessel length and number of vessels , we calculated an angiogenesis index ( ai ) , as previously described by tamargo et al . .
briefly , the number of new vessels associated with each vegf polymer was given a numbered score : 0 = no vessels , 1 = < 10 vessels , 2 = 10 vessels with a visible iris , and 3 = 10 vessels with no visible iris .
neovessel length was assessed as distance from the limbus to the leading edge of the new vessels in millimeters ( mm ) .
ai was then calculated as ( 1)ai = vessel  length  mmvessel  number  score.given that the greatest vessel length achievable was 2  mm ( distance to the vegf polymer from the limbus ) and the highest vessel number score was 3 , the ai ranged from 0 to 6 .
after fixation in formalin , corneas were removed and cut in half at the midline , one vegf polymer pellet in each half .
all cornea halves were embedded in paraffin with the cornea - sclera border facing down , sectioned , and stained with hematoxylin and eosin ( h&e ) .
the extent of neovascularization was analyzed and photographed using digital , light microscopy ( bx41 , olympus , japan ; diagnostic instruments , inc . , sterling heights , mi ) .
total sample size was 32 with measurements of neovascularization taken on days 5 , 8 , 12 , and 15 .
vessel length and ai values recorded throughout the observation period were analyzed for statistical significance using a repeated measures 2-way analysis of variance ( 2-way anova ) .
post hoc analysis was performed using the bonferroni test to correct for multiple comparisons ( graphpad prism 5.0 , ca ) .
the representative images taken on days 0 , 8 , and 15 before and after treatment with bv and fce are shown in figure 2 .  
while ns control developed corneal vessels nearly 2  mm length , all treatment groups had a statistically significant decrease in vessel length compared to ns on all observation days ( p < 0.001 ) .
fce 2 significantly decreased vessel length more than fce 20 on days 12 and 15 ( p < 0.05 ) . bv inhibited vessel length significantly more than fce 2 on days 12 and 15 ( p < 0.05 ) . bv decreased vessel length significantly more than fce 20 on days 8 , 12 , and 15 ( p < 0.01 ) .
ai provided a more comprehensive analysis of the neovascularization as it reflects a more direct assessment of neovessel density .
all treatment groups showed a significant decrease in the ai compared to ns on all observation days ( p < 0.001 ) .
fce 2 , when compared to ns , decreased ai by a factor of 8.0-fold , 7.0-fold , 8.8-fold , and 6.8-fold on days 5 , 8 , 12 , and 15 , respectively .
fce 20 , when compared to ns , decreased ai by a factor of 68-fold , 2.7-fold , 3.6-fold , and 2.4-fold at days 5 , 8 , 12 , and 15 , respectively .
bv decreased ai compared to ns by a factor of 138-fold on day 15 , with no vessel growth at days 5 , 8 , and 12 . a significant difference in ai was not found between bv and fce 2 during the study period , although bv appeared to have a nonsignificant qualitative improvement over fce 2 during the study period .
bv was found to have a significantly smaller ai than fce 20 on days 8 , 12 , and 15 ( p < 0.01 ) . by day 15 ,
ai of fce 2 was significantly smaller than fce 20 ( p < 0.01 ) .
the cornea sections within the ns group showed the greatest corneal neovascularization with a relative increase in both vessel number and vessel circumference when compared with the treatment groups ( figure 5 ) .
the neovascularization present in the bv group was primarily isolated to the superficial stroma and showed a relative decrease in circumference .
fce corneal sections revealed decreased neovascularization , with decreased vessel circumference , when compared with ns but a greater amount than corneas within the bv group .
there was no notable histological difference between the neovascularization that occurred in the fce 2 group and the fce 20 group .
subconjunctival injection of fce was found to be well tolerated and to significantly decrease corneal neovascularization in a rabbit model of vegf - induced neovascularization .
although bv inhibited the corneal neovascularization best in this model , no statistical difference was identified between fce 2 and bv .
fce 20 had an early decrease in neovascularization noted on day 5 , but this benefit was less than that noted by fce 2 on day 8 .
no corneal ulcers , edema , infection , or conjunctival necrosis was noted as a result of either the subconjunctival injection of fce or bv .
the purpose of this study was as a proof of concept to determine the safety and relative efficacy of fce in inhibiting corneal neovascularization .
we chose to use a vegf - induced corneal neovascularization model and as a positive control bv , a monoclonal antibody to vegf , was used . in the short time course of this study
human fce , however , was found to be noninferior to bv , representing an important finding .
fce may be more effective than bv on a multicytokine angiogenesis model given its known multitargeted antiangiogenic effect .
fce 2 was found to outperform the higher concentration fce 20 by the completion of the experiment on day 15 .
initially , fce 20 had an impressive decrease in neovascularization on day 5 , but this effect had weaned by day 15 .
this finding had been described by celik et al . , who noted the antitumor activity of endostatin to have a biphasic dose - response curve [ 22 , 27 ] .
efficacy is found to proportionally increase until an optimal dose is reached with higher doses resulting in decreased response .
a biphasic dose - response curve has been noted in studies of other antiangiogenic cytokines such as interferon- ( ifn- ) [ 27 , 28 ] and the diabetes medication rosiglitazone , a known tumor cell angiogenesis inhibitor .
further dose response analysis will need to be performed within the corneal neovascularization model to fully characterize the dose - response relationship .
the first limitation comes as a result of the corneal neovascularization model selected that allows for the controlled release of vegf and does not entirely mimic the natural proangiogenic environment in which corneal neovascularization develops within a damaged cornea .
additional studies utilizing other models such as the mechanical limbal injury - induced corneal neovascularization model , alkali - induced corneal neovascularization model [ 31 , 32 ] , and the corneal micropocket tumor implantation model   should be considered in future experiments .
secondly , the study was not designed to determine the most optimal dose of fce or the cause of the biphasic dose response .
additional studies to determine the mechanism of inhibition and establish a complete dose - response curve are warranted
. a third limitation of the current study is that long - term efficacy of fce was not assessed .
further preclinical research would need to be completed prior to pursuing human trials into fce as a potential novel inhibitor of corneal neovascularization .
fce is a novel antiangiogenic compound shown to significantly inhibit new vessel growth in a rabbit corneal neovascularization model .
lower concentration fce 2 exhibited better inhibition than fce 20consistent with previous fce studies referencing a biphasic dose - response curve .
the fce 2 concentration was found to be noninferior to bv in this vegf model .
because of its known multitargeted , antiangiogenic properties , fce holds great promise for future clinical efficacy .
further studies are necessary to elucidate the clinical potential and optimal dosing of this novel inhibitor of corneal neovascularization .
 we assessed the antiangiogenic effects of subconjunctival injection of fc - endostatin ( fce ) using a human vascular endothelial growth factor - induced rabbit corneal neovascularization model . 
 angiogenesis was induced in rabbit corneas through intrastromal implantations of vegf polymer implanted 2  mm from the limbus . 
 nzw rabbits were separated into groups receiving twice weekly subconjunctival injections of either saline ; 25  mg / ml bevacizumab ; 2  mg / ml fce ; or 20  mg / ml fce . 
 corneas were digitally imaged at 5 time points . an angiogenesis index ( ai ) was calculated ( vessel length ( mm )  vessel number score ) for each observation . 
 all treatment groups showed a significant decrease in the vessel length and ai compared to saline on all observation days ( p < 0.001 ) . by day 15 , fce 2 inhibited angiogenesis significantly better than fce 20 ( p < 0.01 ) . 
 there was no significant difference between fce 2 and bv , although the values trended towards significantly increased inhibition by bv . 
 bv was a significantly better inhibitor than fce 20 by day 8 ( p < 0.01 ) . 
 fce was safe and significantly inhibited new vessel growth in a rabbit corneal neovascularization model . 
 lower concentration fce 2 exhibited better inhibition than fce 20 , consistent with previous fce studies referencing a biphasic dose - response curve . 
 additional studies are necessary to further elucidate the efficacy and clinical potential of this novel angiogenesis inhibitor . 
1. Background
2. Methods
3. Results
4. Discussion
5. Conclusions