angiogenesis , the formation of new vessels from existing vessels , plays an important role in pathological conditions in various organs .
pathological angiogenesis in the eye is the most common cause of blindness in all age groups .
retinopathy of prematurity ( rop ) occurs in children , diabetic retinopathy ( dr ) in young adults , and age - related macular degeneration ( amd ) in the elderly .
it is important to understand the mechanism of underlying pathological neovascularization to identify new targets to treat these diseases . vascular endothelial growth factor ( vegf )
is thought to be the major angiogenesis factor in rop , dr , and amd .
recently , some evidence indicates that chronic inflammation is also implicated in the pathogenesis of retinal neovascularization [ 4 , 5 ] .
the high - mobility group box-1 ( hmgb1 ) protein was initially discovered as a nuclear chromatin - binding protein that stabilizes nucleosome formation and facilitates transcription .
hmgb1 can be actively secreted by various cell types , including activated monocytes and macrophages , and endothelial cells , after inflammatory stimuli [ 7 , 8 ] .
extracellular hmgb1 functions as a proinflammatory cytokine [ 6 , 9 ] and exhibits angiogenic effects [ 10 , 11 ] .
hmgb1 signals through the receptor for advanced glycation end products ( rage ) leading to the activation of the transcription factor nuclear factor kappa b ( nf-b ) and induces the expression of various leukocyte adhesion molecules proinflammatory cytokines , chemokines , and angiogenic factors [ 6 , 9 ] . in a previous report
, increased levels of hmgb1 were found in vitreous samples from patients with proliferative dr , and hmgb1 expression was upregulated in the retinas of diabetic mice .
ethyl pyruvate ( ep ) is derived from pyruvate by the addition of an aliphatic ester group and is more stable and safer than pyruvate in inhibiting the generation of reactive oxygen species ( ros ) and inflammation .
moreover , ep inhibits tumor angiogenesis and intracerebral hemorrhage - induced angiogenesis [ 16 , 17 ] .
however , the pathogenic role of hmgb1 and the effect of its inhibitor , ep , in pathological retinal neovascularization have remained uncertain . in the present study
, we examined whether ep has a preventive effect against pathological retinal neovascularization and inhibits hmgb1 expression in a mouse model of oxygen - induced retinopathy ( oir ) .
oir was induced in mouse ( c57bl/6 ) pups according to smith et al .   with some modifications .
briefly , postnatal day 7 ( p7 ) mice , with their nursing mothers , were exposed to hyperoxia ( 75  2% oxygen ) for five days ( p7p11 ) to produce retinal vasoobliteration and then returned to normoxia ( room air ) for five days ( p12p16 ) to induce ischemic retinal neovascularization . at p12 , after the pups were exposed to 75% oxygen , they were randomly assigned to three groups : ep-50 ( 50  mg / kg / day ) , ep-100 ( 100  mg / kg / day ) , and the oir .
the normal group was maintained in room air from birth until postnatal day 17 ( p17 ) .
louis , mo , usa ) was diluted with hartmann 's solution ( 6.0  g nacl , 0.3  g kcl , 0.2  g cacl22h2o , and 3.1  g c3h5nao3/l , choongwae pharma , republic of korea ) .
the pups were injected in 100  l intraperitoneally once a day for five days . in the normal group ,
hartmann 's solution was injected . at p17 , after five days of intraperitoneal injection , the mice were anesthetized and sacrificed .
all of the experiments were approved by the korean institute of oriental medicine institutional animal care and use committee . at p17 ,
the mice were deeply anesthetized and then 0.1  ml of phosphate - buffered saline ( pbs ) containing 5  mg of fluorescein - dextran ( fd40s , sigma , st .
the retinas were dissected , flat mounted onto glass slides , and viewed by fluorescence microscopy ( bx51 , olympus , tokyo , japan ) .
quantification of the central nonperfused area of the retina was performed using the imagej software ( nih , md , usa ) .
flat - mounted retinas were fixed in 4% paraformaldehyde for 3  h at room temperature .
the retinas were washed with pbs and then incubated for 3  h on an orbital shaker at room temperature with 5% triton x-100 and 1% bsa .
the retinas were washed with pbs and incubated overnight at 4c with bandeiraea simplicifolia isolectin b4 ( 1  :  50 , sigma - aldrich , st .
louis , mo ) diluted in pbs . the retinas were washed with 0.05% tween 20 in pbs followed by incubation with streptavidin tritc ( 1  :  500 , serotec , oxford , uk ) for 4  h at 37c .
the samples were added in extraction buffer , 20  mm tris hcl buffers ( ph 4 ) with 2% sds , incubated on ice for 5  min , and mixed by vortexing then boiled at 100c for 20  min followed by an optional incubation at 80c for 2 hours . after protein extraction ,
any remaining unsolubilized material was pelleted at 14000  g for 20  min , and protein concentration of total protein extracted was determined by the bca protein assay ( pierce chemicals co. , rockford , il , usa ) .
the protein was separated by sds - polyacrylamide gel electrophoresis and transferred to pvdf membrane ( biorad , ca , usa ) .
membrane was probed with anti - hmgb1 antibody ( abcam , ma , usa ) and anti -actin antibody ( sigma , mo , usa ) , and then the immune complexes were visualized with an enhanced chemiluminescence detection system ( amersham bioscience , nj , usa ) .
the eyes were removed and fixed with davidson 's solution at room temperature for 24  h and embedded in paraffin .
the paraffin sections were deparaffinized , hydrated with water , and stained with hematoxylin and eosin . for immunohistochemical hmgb1 staining
, paraffin sections were deparaffinized , rinsed with 3% hydrogen peroxide , and boiled in citrate buffer ( ph 6.0 ) for 15  min .
these sections were washed with pbs , blocked with cas - block ( invitrogen , ca , usa ) for 30  min , and subsequently incubated overnight at 4c with a rabbit anti - hmgb1 antibody ( 1  :  1000 , epitomics , ca , usa ) .
after washing with pbs , the sections were incubated with polymer peroxidase - conjugated rabbit anti - igg for 30  min at room temperature and were visualized using a 3,3-diaminobenzidine tetrahydrochloride ( immpact dab , vector , ca , usa ) solution and hematoxylin ( vector , ca , usa ) .
the data were expressed as the mean  se and analyzed by one - way analysis of variance ( anova ) followed by tukey 's multiple comparison test or by an unpaired student 's t - test using graphpad prism 6.0 software ( graphpad , ca , usa ) .
hmgb1 has been recognized as a proinflammatory cytokine and more recently as a proangiogenic factor [ 6 , 9 ] .
we therefore determined the expression levels of hmgb1 and its distribution in the retina of oir mice by immunohistochemistry .
first , retinal neovascularization was qualitatively analyzed using fluorescein angiography and quantitatively analyzed by counting neovascular tufts using isolectin b4 and h&e staining .
the oir mice showed a characteristic loss of central retinal vessels by p12 , followed by hypoxia - induced regeneration of the central vascular plexus and the development of preretinal neovascularization . in the oir group ,
the neovascular response occurred predominantly at the junction between the nonperfused retina and perfused retina .
the retinas of oir mice had an area of multiple neovascular tufts ( figures 1 and 2 ) .
in western blot analysis and immunohistochemical staining for hmgb1 , we found that hmgb1 was increased in the retinas of oir mice compared with the control group ( figures 3 and 4 ) .
hmgb1 is highly expressed in ganglion cells , inner nuclear layers , outer nuclear layer , and retinal vasculatures , and hmgb1 levels were higher in the inner nuclear layer than the outer nuclear layer in oir mice ( figure 4 ) .
elevated expression of hmgb1 was detected in the nuclei as well as in the cytoplasm in oir mice .
these results suggest that hmgb1 is highly produced and translocates into the cytoplasm in retina of oir mice .
it was previously reported that a dramatic alteration of retinal gene expression occurred in oir mice , and a high level of retinal hmgb1 expression might be related to active gene transcription .
thus , an increase in hmgb1 might be related to active angiogenic - related gene transcription .  
, we examined whether ep has a preventive effect against pathological retinal neovascularization and inhibits hmgb1 expression . to test whether pharmacological inhibition of hmgb1 decreases pathological retinal neovascularization ,
the oir mice induced the formation of neovascular tufts that penetrate the internal limiting membrane of the retina ( figure 2 ) .
five days after the injection ( at p17 ) , there were dose - dependently fewer neovascular tufts in the ep - injected eyes compared to the oir group ( figure 4 , p < 0.05 ) .
moreover , the treatment with ep significantly reduced retinal hmgb1 expression in oir mice ( figures 3 and 4 ) .
it thus seemed that hmgb1 in the retina of oir mice has a proangiogenic role and ep has an antiangiogenic effect via inhibition of the production and release of hmgb1 .
in addition , the retinas of oir mice had an area of nonperfusion in the center , tortuous vessels ( figure 1 ) . however , the ep ( 100  mg / kg)-treated mice had a significantly smaller nonperfused area compared to controls ( figure 1 , p < 0.01 ) .
retinal neovascularization ( rnv ) is a major cause of blindness associated with ischemic retinopathy [ 2 , 23 ] .
oir is a well - established animal model of proliferative ischemic retinopathy . in the oir model ,
revascularization of the vasoobliteration ( vo ) area has progressed farther and pathologic neovascularization formation is at its maximum at p17 .
the vo area is then fully revascularized in all of the layers , and neovascularization is completely resolved at p25 . in this study , to verify the pathogenic role of hmgb1 in retinal neovascularization , we determined the expression levels of hmgb1 and its distribution in the retina of oir mice and examined whether ep , a well - known hmgb1 inhibitor , could prevent this retinal neovascularization in oir mice .
we showed that nuclear and cytoplasmic overexpression of hmgb1 and subsequent angiogenesis occurred in oir mice .
however , the treatment with ep dramatically promoted stable vascular growth and blocked retinal pathologic neovascularization .
based on these results , it is likely that the retinal pathogenic angiogenesis that occurs in ischemic retinopathy could also be linked to increases in hmgb1 expression .  
several angiogenic growth factors and cytokines are involved in the pathogenesis of retinal neovascularization [ 2527 ] . a critical role for hmgb1 in angiogenesis
has been recently suggested by wake et al . who reported that histidine - rich glycoproteins block hmgb1-heparin complex - induced vessel sprouting in matrigel plugs .
hmgb1 signals through the rage leading to activation of the transcription fact or nuclear factor kappa b ( nf-b ) and induces the expression of various angiogenic factors [ 9 , 29 ] .
administration significantly increases the levels of growth factors , including vegf , released by cultured human cardiac fibroblasts .
although concrete evidence of hmgb1-induced retinal angiogenesis is lacking , our findings suggest that hmgb1 might provide the mechanistic link between ischemic retinopathy and pathogenic angiogenesis .  
in oir , ros are increased in the retina and nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase is activated , causing apoptosis in endothelial cells , which contributes to avascular retinas .
nadph oxidase activation is exacerbated , contributing to angiogenic blood vessel growth into the vitreous .
ros are increased in the retina , which directly correlates with vegf expression and angiogenesis .
in addition , oxidative stress in various cells is induced in ischemic injury , and then hmgb1 is actively secreted into the extracellular environment under these conditions .
hydrogen peroxide ( h2o2 ) and the loss of superoxide dismutase 1 ( sod1 ) mediated oxidative stress promote cytosolic hmgb1 expression and extracellular release .
although we did not test the retinal oxidative status in our oir mice , these findings suggest that increased retinal hmgb1 expression might be mediated through retinal ros generation . in this study
, we showed that ep treatment prevents the overexpression of nuclear and cytoplasmic hmgb1 and retinal pathogenic angiogenesis in oir mice .
ep has salutary effects in lethal sepsis and systemic inflammation , hemorrhagic shock , and stroke   and pressure - induced retinal damage   and ameliorates murine colitis   and renal ischemia and reperfusion injury .
the pharmacological effects of ep include amelioration of redox - mediated damage to cells and tissues and the scavenging of ros [ 16 , 42 ] .
ep regulates the activation of the expression of inflammatory proteins such as il-6 and tnf- and the inflammatory transcription factor nf-b [ 43 , 44 ] .
this pharmacological agent has been shown to interfere specifically with hmgb1 released from the nucleus into the extracellular space . in a previous study
, ep prevented the increase of hmgb1 mrna expression after raw264.7 cells were stimulated with lps .
although ep is expected to exert an antioxidant effect , ep could be a potent hmgb1 inhibitor .
this observation suggests that the antiangiogenic effect of ep is attributable , at least in part , to not only its antioxidative but also hmgb1-inhibitory properties .
therefore , to confirm a proangiogenic role of hmgb1 , we also performed a similar animal study using a selective inhibitor of hmgb1 , glycyrrhizin ( gl ) .
gl is a triterpenoid saponin glycoside of glycyrrhizic acid and its mechanism of action is different from ep .
ep inhibits the expression and cytoplasmic release of hmgb1 but do not bind directly to hmgb1 and thus can not block its extracellular functions .
in contrast , gl does not interfere with hmgb1 release , but directly inhibit its extracellular cytokine activities .
gl treatment also significantly reduced the area of neovascularization in retinas compared with oir ( see supplementary data in supplementary material available online at http://dx.doi.org/10.1155/2013/245271 ) .
this observation also strongly supports the proangiogenic role of hmgb1 in oir mice . in conclusion
, our study shows for the first time that ep inhibits the retinal pathogenic angiogenesis induced by ischemic retinopathy in oir mice .
in addition , hmgb1 overexpression induced by ischemic retinopathy was significantly inhibited by treatment with ep .
these observations suggest that ep acts through an anti - hmgb1 mechanism to protect against retinal pathogenic angiogenesis . taken together ,
these results indicate that treatment with ep could be a valuable therapeutic approach in the treatment or prevention of ischemic retinopathy .
 retinal pathogenic angiogenesis in the eyes is a causative factor in retinopathy of prematurity , diabetic retinopathy , and age - related macular degeneration . 
 this study was designed to examine the pathogenic role of the high - mobility group box-1 ( hmgb1 ) protein and the inhibitory effect of ethyl pyruvate ( ep ) , a well - known antioxidant substance , in retinal pathogenic angiogenesis in mice with oxygen - induced retinopathy ( oir ) , one of the animal models of proliferative ischemic retinopathy . 
 the oir mouse model was used for our in vivo studies . 
 the mice were exposed to 75% oxygen from postnatal day 7 ( p7 ) to p11 , after which the mice were brought to room air and intraperitoneally injected with ep ( 50  mg / kg , or 100  mg / kg ) for five days . at p17 , the mice were perfused with fluorescein isothiocyanate - dextran , and flat - mounted retinas were used to measure nonperfused and neovascular tufts . in oir mice , 
 an intraperitoneal injection of ep reduced the nonperfused retinal area in the treatment group and significantly reduced the retinal neovascular tufts . 
 in addition , ep inhibited the overexpression of hmgb1 in the retinas of oir mice . 
 these data suggest that ep could serve as an innovative pharmaceutical agent to prevent retinal neovascularization through inhibiting hmgb1 expression . 
1. Introduction
2. Materials and Methods 
3. Results
4. Discussion 