diabetic retinopathy ( dr ) is a leading cause of visual impairment and blindness in developed countries .
the decrease in vision is due to diabetic macular edema ( dme ) and proliferative diabetic retinopathy ( pdr ) [ 1 , 2 ] .
the control of the blood glucose level , vitreous surgery , and photocoagulation are the major treatments used to prevent dr from progressing to the pdr stage .
currently , intravitreal injections of antivascular endothelium growth factor ( vegf ) or steroids have become the primary therapy for dme [ 35 ] .
however , the use of a single therapeutic agent is not effective in all cases , and additional treatments or different agents are needed . the blood retinal barrier ( brb )
consists of two anatomical parts ; the inner brb is located within the endothelial cells of the retinal capillaries , and the outer brb is located between the retinal pigment epithelial ( rpe ) cells . an intact brb is required for an efficient and regulated control of fluids in the subretinal space and for the maintenance of healthy rpe and retinal cells .
a breakdown of the outer brb results in an increase in the paracellular permeability between the rpe cells , and the leakage can cause retinal edema .
the tight junctions ( tjs ) of the rpe cells are intercellular junctions located at the apical ends of the rpe cells , and they are integral structural components of the brb .
tjs are made up of three tj related proteins , for example , the zonula occludens-1 ( zo-1 ) , occludin , and claudin .
recent studies have shown that interactions between inflammatory cells and retinal cells are critical for the development of intraocular neovascularization [ 1012 ] .
the results of these studies also demonstrated that , in eyes with pdr , an elevation of vegf was significantly correlated with the levels of several cytokines including interleukin- ( il- ) 6 , il-8 , and the monocyte chemoattractant protein-1 ( mcp-1 ) .
another study showed that the levels of these cytokines were also strongly correlated with each other which suggest that there are common pathways involved in the inflammatory processes .
in addition , various cytokines have been shown to be related to the maintenance of the conformation of the tj proteins .
lecithin - bound iodine ( lbi , jolethin , daiichi pharmaceutical co. , tokyo , japan ) has been used clinically to reduce the antigen - induced immune responses in children with bronchial asthma .
lbi acts on the peripheral blood mononuclear cells and downregulates the il-4-induced ige synthesis , which suggests that lbi have anti - inflammatory properties [ 15 , 16 ] . in patients with eye diseases ,
lbi has been used for the absorption of retinal or vitreous bleeding , vitreous opacities , and improvement of central serous choroidopathy .
the purpose of this study was to determine whether lbi will alter the integrity of the tjs of arpe-19 cells in culture .
lbi ( jolethin , daiichi pharmaceutical co. , tokyo , japan ) was dissolved in distilled water and diluted to the appropriate concentration for each experiment .
an earlier study showed that lbi solutions contain 48.250.3% lecithin - iodine , approximately 10% free lecithin , and 40% phosphatidylinositol .
rabbit polyclonal anti - zo-1 antibody ( sc-10804 ) was purchased from santa cruz biotechnology ( santa cruz , ca ) , and alexa 594 anti - rabbit igg was purchased from invitrogen ( molecular probs , eugene , or ) .
rabbit anti - mcp-1 antibody ( # 2029 ) was purchased from cell signaling technology ( danvers , ma ) and rabbit anti - eotaxin ( ccl-11 ) antibody ( ab133604 ) was purchased from abcam ( cambridge , ma ) .
cells from a human retinal pigment epithelium cell line , arpe-19 , were grown in dulbecco 's modified eagle 's medium / ham 's f-12 supplemented with 10% fetal bovine serum ( fbs , hyclone laboratories , inc . ,
logan , ut ) , 50  units / ml penicillin , and 50  g / ml streptomycin in an air-5% co2 atmosphere with constant humidity .
prior to the experiments , cells were placed in serum - free media and pretreated with 50  g / ml of lbi or 250500  pg / ml of antibody ( anti - mcp-1 antibody and anti - chemokine ( c - c motif ) ligand-11 ( ccl-11 ) antibody ) for 24  h. to mimic hypoxic conditions , arpe-19 cells were incubated for 4  h with 100  m of cocl2 ( sigma , st .
louis , mo ) , which is a chemical hypoxia - inducing agent . after the incubation ,
optimum concentration of lbi was defined as 50  g / ml because over 100  g / ml of lbi seems to be toxic ( data not shown ) . for the immunofluorescence studies , cells
were grown to a density of 2  10 cells / ml on 12  mm cover slips and fixed in 10% tricarboxylic acid for 10  min at 4c .
they were then treated with 0.5% triton x-100 for 15  min . to detect the presence of zo-1 , the cultured rpe cells were exposed to rabbit polyclonal anti - zo-1 antibody as the primary antibody and alexa 594 anti - rabbit igg as the secondary antibody .
samples were examined and photographed with a fluorescence microscope ( bz-9000 ; keyence , osaka , japan , and eclipse 50i , nikon , tokyo , japan ) , and the intensity of fluorescence was quantified with a bz - ii analyzer ( keyence ) .
the levels of the bioactive molecules in the conditioned medium were determined by human il-6 , il-8 , and mcp-1 elisa kits ( r&d systems , minneapolis , mn ) and ccl-11 elisa kit ( biosensis , thebarton , south australia , australia ) .
all experiments were repeated at least three times and values are presented as the means  standard deviations .
data were analyzed by two - way nonrepeated analysis of variance ( anova ) followed by bonferroni post hoc tests for the comparison of the means .
we first determined whether the lbi pretreatment affected the changes in the conformation of the tj proteins caused by the hypoxic stress induced by cocl2 .
immunofluorescence microscopy showed that there appeared to be a disruption of tjs with cocl2 treatment ( figures 1(a ) and 1(b ) ) .
when the arpe-19 cells were pretreated with lbi before the addition of cocl2 , the disruption of the tight junctions was not detected ( figures 1(c ) and 1(d ) ) .
the signal intensity measurements showed that there was a significant decrease with the cocl2 addition compared to the control .
but there was no significant change in the lbi pretreated group ( 29244.6  2981.2 in controls ; 5787.7  4126.4 with cocl2 ; and 27189.0  11231.1 with cocl2 after lbi pretreatment ; p < 0.05 , nonrepeated anova , n = 5 , figure 1(e ) ) . because disruption of tjs is detected as a decrease of signal intensity , these results indicate that lbi pretreatment can protect the tjs of the outer brb from hypoxic stress .
because lbi pretreatment protected the conformation of the tj proteins from hypoxic stress , we hypothesized that lbi will block different inflammatory molecules that are secreted from cells during hypoxia .
the results of the elisa measurements of the conditioned culture media ( n = 5 ) indicated that il-8 was not significantly changed after the addition of cocl2 or after pretreatment with lbi ( control , 6.9  0.1 ; with cocl2 , 6.8  0.1 ; and with cocl2 after lbi pretreatment , 6.9  0.1  pg / ml ; figure 2(a ) ) .
although the level of il-6 was increased after addition of cocl2 which was decreased by lbi pretreatment , these changes were not significant ( control , 10.4  1.6 ; cocl2 addition , 15.0  6.7 ; and cocl2 addition after lbi pretreatment , 13.3  7.6  pg / ml ; figure 2(b ) ) .
the level of mcp-1 increased significantly after the addition of cocl2 , and it was significantly decreased after the addition of cocl2 after lbi pretreatment ( control , 279.7  68.3 ; with cocl2 , 340.8  43.3 ; and with cocl2 after lbi pretreatment , 182.6  23.8  pg / ml , p < 0.05 ; nonrepeated anova ; figure 2(c ) ) .
a similar tendency was observed for ccl-11 ( control , 12.5  6.1 ; with cocl2 , 15.2  12.9 ; and with cocl2 after lbi pretreatment , 5.46  1.9  pg / ml ; p < 0.05 ; nonrepeated anova ; figure 2(d ) ) .
these results indicated that there is an increase of the mcp-1 and ccl-11 secretion under hypoxic stress , and all are suppressed by lbi pretreatment .   to confirm that lbi effectiveness is related to suppression of mcp-1 or ccl-11
, cells were pretreated with anti - mcp-1 or anti - ccl-11 antibody for 24  hrs .
the tj disruption from hypoxic stress was reduced with pretreatment ( figures 3(e ) and 3(f ) ) .
these results indicated that both mcp-1 and ccl-11 were important to protect tjs from hypoxic stress and support the effectiveness of lbi .
vegf is a major antigen factor and is biogenic permeability factor which can increase the vascular permeability endothelial cell - cell junctions . finally , to confirm that lbi pretreatment can protect tjs from direct vegf induced stress , after lbi pretreatment
but after pretreatment of lbi , this disruption was decreased which indicates that lbi can also protect tjs from vegf induced damage .
but there still remain patients who do not respond to the anti - vegf therapy   and who had a reduction of their vision even after therapy .
it must also be noted that the cost of anti - vegf therapy is quite high and continuous injections to control the dme can become an economic issue .
combination therapies have also been proposed that can enhance the efficacy   and may minimize the cost of anti - vegf therapies .
for example , photocoagulation can extend the interval of anti - vegf injections resulting in a reduction in the number of injections [ 21 , 22 ] .
if new drugs or treatments can be approved , this will make it easier and less expensive to use combination therapies .
moreover , adjunctive therapies that include oral kallidinogenase could also be used for combined therapy [ 23 , 24 ] .
our results showed that the hypoxic stress induced by exposure of arpe-19 cells to cocl2 caused a disruption of the tight junctions and that lbi pretreatment can protect the tjs from the hypoxic stress .
hypoxic stress also enhanced the secretion of mcp-1 and ccl-11 , and this enhancement can be suppressed by lbi pretreatment .
hypoxia has been reported to produce alterations in the tight junction proteins that are correlated with the vascular permeability increases .
the integrity of the brb is dependent on tj - associated proteins such as occludin , claudin , and zo-1 .
the function of zo-1 is to link the transmembrane protein , occludin , to the actin cytoskeleton . under hypoxic conditions ,
the phosphorylation of zo-1 is enhanced which is related to the damage of the tight junctions .
mcp-1 is associated with a breakdown of the brb [ 3032 ] , and it also induces a disruption of the tjs by the caveolae that are dedicated to the internalization of the tj proteins , such as zo-1 .
because mcp-1 is also increased in the vitreous of patients with dr , it probably plays an important role in the progression of dr and dme . in the photocoagulation - induced mouse model of retinal neovascularization , it was reported that an increase in mcp-1 contributes to the postischemic inflammation and dr progression [ 34 , 35 ] .
mcp-1 has been recently postulated to have a direct effect on angiogenesis even though there is no link with macrophage recruitment .
ccl-11 also promotes the recruitment of vascular endothelial cells and relates the conformation of the tight junctions .
interestingly , there is also an increase of ccl-11 in the proliferative membranes obtained from eyes with pdr .
so these cytokines relate to zo-1 and maintain tight junctions on diabetic retina . in the photocoagulation - induced retinal neovascularization or light - exposed mouse models ,
the levels of not only mcp-1 but also ccl-11 are increased in the rpe [ 3941 ] .
these increases were related to the remodeling of f - actin that occurred after light damage and caused proinflammatory changes in the rpe cells .
these findings support our results that showed that the hypoxia - induced stress affected the expression of cytokines especially mcp-1 and ccl-11 .
the suppression of these cytokines by lbi leads to the protection of these cells from disruption of the tight junctions .
steroids are also commonly used for dme therapy because their anti - inflammatory property helps to protect the tissues from neovascularization or vascular permeability increases .
similarly , the effect of lbi on the suppression of the postischemic inflammation may be an additional contributor when used as either a primary or adjunctive dme therapy . mainly in japan and china ,
lbi has been approved for the treatment of various retinal diseases including central serous chorioretinopathy , vitreous hemorrhage , or vitreous opacity for a long time .
in addition , both lecithin and iodine are commonly used in food materials indicating the safety of lbi .
thus , the possibility exists that lbi can be even used as a safe adjunctive therapy with an anti - vegf drug to treat dme .
further investigations into the use of lbi in conjunction with anti - vegf therapy need to be undertaken to determine the effectiveness of lbi in preventing the development or progression of dme .
our findings showed that lbi pretreatment reduced the levels of mcp-1 and ccl-11 and blockage of them also results in protection of tjs .
however , no significant changes were detected in the levels of il-6 and il-8 , which are the major cytokines related to neovascularization . in ischemic
retinas , il-8 causes increases in the vegf and mcp-1 levels but it is dependent on tnf- .
because il-8 has been shown to be secreted from either monocytes or macrophages   and because our experiments were limited to arpe-19 cells in culture , there is the possibility that the results may not hold under in situ conditions . though we observed that vascular permeability improved after oral lbi administration for diabetic mice model ( data not shown ) , it is not clear whether this is the result of hypoxia improvement the same as we show here using arpe-19 .
in addition , lbi pretreatment can also protect tjs from direct vegf induced damage which results in tj disruption .
further investigations will need to be undertaken to definitively explain these phenomena . in conclusion ,
our data demonstrate the therapeutic potential of lbi for protecting the integrity of the tight junctions from hypoxia - induced stress .
these data suggest a new aspect on the clinical use of lbi in the treatment of dme .
 
 aim . we investigated 
 whether lecithin - bound iodine ( lbi ) can protect the integrity of tight junctions of retinal pigment epithelial cells from hypoxia . method . 
 cultured human retinal pigment epithelial ( arpe-19 ) cells were pretreated with lbi . 
 to mimic hypoxic conditions , cells were incubated with cocl2 . 
 we compared the integrity of the tight junctions ( tjs ) of control to cells with either lbi alone , cocl2 alone , or lbi + cocl2 . 
 the levels of cytokines in the conditioned media were also determined . 
 results . 
 significant decrease in the zonula occludens-1 ( zo-1 ) intensity in the cocl2 group compared to the control ( 5787.7  4126.4 in cocl2 group versus 29244.6  2981.2 in control ; average  standard deviation ) . 
 but the decrease was not significant in the lbi + cocl2 ( 27189.0  11231.1 ) . 
 the levels of monocyte chemoattractant protein-1 ( mcp-1 ) and chemokine ( c - c motif ) ligand 11 ( ccl-11 ) were significantly higher in the cocl2 than in the control ( 340.8  43.3 versus 279.7  68.3  pg / ml for mcp-1 , and 15.2  12.9 versus 12.5  6.1  pg / ml for ccl-11 . 
 with lbi pretreatment , the levels of both cytokines were decreased to 182.6  23.8 ( mcp-1 ) and 5.46  1.9  pg / ml for ccl-11 ) . 
 blockade of mcp-1 or ccl-11 also shows similar result representing tj protection from hypoxic stress 
 . conclusions . 
 lbi results in a protective action from hypoxia . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion