six- to 8-week - old male balb / c and c57bl/6 mice ( taconic farms , germantown , ny , usa ) were used in the experiments , and mice were anesthetized using a mixture of ketamine , xylazine , and acepromazine ( 50 mg , 10 mg , and 1 mg / kg body weight , respectively ) for each surgical procedure .
all mice were treated according to the arvo statement for the use of animals in ophthalmic and vision research and all protocols were approved by the animal care and use committee , university of california , berkeley .
orthotopic corneal transplantation was performed between fully mismatched c57bl/6 ( donors ) and balb / c ( recipients ) , as reported previously .
basically , donor central cornea was marked with a 2-mm - diameter microcurette ( katena products , inc . ,
denville , nj , usa ) and excised with vannas scissors ( storz instruments co. , san dimas , ca , usa ) .
the recipient graft bed was similarly prepared with a 1.5-mm - diameter microcurette and the donor button was secured in recipient bed with eight interrupted 11 - 0 nylon sutures ( arosurgical , newport beach , ca , usa ) .
the recipient mice were randomized to receive subconjunctival injections of either hamster anti - mouse itga-9 antibody ( 6.4 g ; kindly provided by toshimitsu uede , md , phd , hokkaido university , hokkaido , japan ) or its isotype control hamster igg ( jackson immunoresearch , west grove , pa , usa ) , as reported previously .
subconjunctival injection was performed twice a week on the day of transplantation and thereafter up to 8 weeks after the surgery .
as described previously , after the transplantation surgery , all eyes were first examined on day 3 and corneal sutures were removed on day 7 .
grafts were evaluated by ophthalmic slit - lamp biomicroscopy twice a week for 8 weeks according to the standard scheme .
basically , the degree of graft opacification was graded between 0 ( clear and compact graft ) to 5 + ( maximal opacity with total obscuration of the anterior chamber ) .
grafts with an opacity score of 2 + or higher after 3 weeks or an opacity score of 3 + or higher at 2 weeks were regarded as rejected .
briefly , whole - mount full - thickness corneas were harvested at 8 weeks after transplantation and fixed in acetone for immunofluorescent staining .
samples were sequentially incubated with purified rabbit anti - mouse lyve-1 ( abcam , cambridge , ma , usa ) antibody and goat anti - mouse itga-9 antibody ( r&d systems , minneapolis , mn , usa ) , which were visualized by fitc - conjugated donkey anti - rabbit and cy3-conjugated donkey anti - goat secondary antibodies ( jackson immunoresearch laboratories ) , respectively .
samples were covered with vector shield mounting medium ( vector laboratories , burlingame , ca , usa ) and examined by an axioimager m1 epifluorescence deconvolution microscope with axiovision 4.8 software ( carl zeiss ag , gttingen , germany ) .
briefly , for lg evaluation , lyve-1 vascular structures were analyzed by imagej software ( http://imagej.nih.gov/ij/ ; provided in the public domain by the national institutes of health , bethesda , md , usa ) .
the lymphatic invasion area was normalized to the total corneal area to obtain a percentage coverage score for each sample .
the total corneal area was measured by outlining the innermost lymphatic vessels of the limbal arcade , and lymphatic invasion area was determined by tracing out the contours of the lyve-1 lymphatic network inside the cornea .
additionally , the cornea was divided into four equal quadrants in reference to the vertical midline passing through the 6- and 12-o'clock positions , and the nasal and temporal quadrants were used for analysis of polarized lymphatic vessel distribution for each sample .
luminal valves also were evaluated and focal itga-9/lyve-1 areas running along the length of the lyve-1 vessels were identified as valves and quantified for each sample .
the percentage scores were obtained by normalizing to the means of control condition that were defined as being 100% .
mann - whitney u test was used to evaluate the statistical significance of the difference between the groups .
the association analysis was performed by the linear mixed model built with the r studio platform ( r studio inc . ,
all other statistical analysis was performed with prism software ( graphpad , la jolla , ca , usa ) ;
six- to 8-week - old male balb / c and c57bl/6 mice ( taconic farms , germantown , ny , usa ) were used in the experiments , and mice were anesthetized using a mixture of ketamine , xylazine , and acepromazine ( 50 mg , 10 mg , and 1 mg / kg body weight , respectively ) for each surgical procedure .
all mice were treated according to the arvo statement for the use of animals in ophthalmic and vision research and all protocols were approved by the animal care and use committee , university of california , berkeley .
orthotopic corneal transplantation was performed between fully mismatched c57bl/6 ( donors ) and balb / c ( recipients ) , as reported previously .
basically , donor central cornea was marked with a 2-mm - diameter microcurette ( katena products , inc . ,
denville , nj , usa ) and excised with vannas scissors ( storz instruments co. , san dimas , ca , usa ) .
the recipient graft bed was similarly prepared with a 1.5-mm - diameter microcurette and the donor button was secured in recipient bed with eight interrupted 11 - 0 nylon sutures ( arosurgical , newport beach , ca , usa ) .
the recipient mice were randomized to receive subconjunctival injections of either hamster anti - mouse itga-9 antibody ( 6.4 g ; kindly provided by toshimitsu uede , md , phd , hokkaido university , hokkaido , japan ) or its isotype control hamster igg ( jackson immunoresearch , west grove , pa , usa ) , as reported previously .
subconjunctival injection was performed twice a week on the day of transplantation and thereafter up to 8 weeks after the surgery .
as described previously , after the transplantation surgery , all eyes were first examined on day 3 and corneal sutures were removed on day 7 .
grafts were evaluated by ophthalmic slit - lamp biomicroscopy twice a week for 8 weeks according to the standard scheme . basically , the degree of graft opacification was graded between 0 ( clear and compact graft ) to 5 + ( maximal opacity with total obscuration of the anterior chamber ) .
grafts with an opacity score of 2 + or higher after 3 weeks or an opacity score of 3 + or higher at 2 weeks were regarded as rejected .
briefly , whole - mount full - thickness corneas were harvested at 8 weeks after transplantation and fixed in acetone for immunofluorescent staining .
samples were sequentially incubated with purified rabbit anti - mouse lyve-1 ( abcam , cambridge , ma , usa ) antibody and goat anti - mouse itga-9 antibody ( r&d systems , minneapolis , mn , usa ) , which were visualized by fitc - conjugated donkey anti - rabbit and cy3-conjugated donkey anti - goat secondary antibodies ( jackson immunoresearch laboratories ) , respectively .
samples were covered with vector shield mounting medium ( vector laboratories , burlingame , ca , usa ) and examined by an axioimager m1 epifluorescence deconvolution microscope with axiovision 4.8 software ( carl zeiss ag , gttingen , germany ) .
briefly , for lg evaluation , lyve-1 vascular structures were analyzed by imagej software ( http://imagej.nih.gov/ij/ ; provided in the public domain by the national institutes of health , bethesda , md , usa ) .
the lymphatic invasion area was normalized to the total corneal area to obtain a percentage coverage score for each sample .
the total corneal area was measured by outlining the innermost lymphatic vessels of the limbal arcade , and lymphatic invasion area was determined by tracing out the contours of the lyve-1 lymphatic network inside the cornea .
additionally , the cornea was divided into four equal quadrants in reference to the vertical midline passing through the 6- and 12-o'clock positions , and the nasal and temporal quadrants were used for analysis of polarized lymphatic vessel distribution for each sample .
luminal valves also were evaluated and focal itga-9/lyve-1 areas running along the length of the lyve-1 vessels were identified as valves and quantified for each sample .
the percentage scores were obtained by normalizing to the means of control condition that were defined as being 100% .
mann - whitney u test was used to evaluate the statistical significance of the difference between the groups .
the association analysis was performed by the linear mixed model built with the r studio platform ( r studio inc . ,
all other statistical analysis was performed with prism software ( graphpad , la jolla , ca , usa ) ; p < 0.05 was considered significant .
we first studied the effect of itga-9 blockade on corneal lg and vg induced by transplantation .
either itga-9 neutralizing body or isotype control was injected subconjunctivally twice a week starting from the surgery date . as demonstrated in figure 1a , following the treatment with the itga-9 blocking antibody , corneal lymphatic vessels contained significantly fewer valves compared with the control condition . summarized data from repetitive experiments are presented in figure 1b ( left ; p < 0.05 ) . however , this treatment had no effect on lg , as shown in figure 1b ( right ) .
our further analysis on the ratio of valve quantity to lymphatic invasion area revealed a significant reduction in this parameter in the treated rather than the control condition ( figure 1c ; p < 0.05 ) .
( a ) representative whole - mount immunostaining images demonstrating significantly fewer valves in the itga-9 blocking antibody  treated cornea in comparison with isotype control  treated cornea .
( b ) comparative quantification on lymphatic valves and lymphatic vessel invasion area in control and treatment conditions . anti 
the experiment was repeated twice with seven mice in control and eight mice in treatment group . * p < 0.05 ; n.s . , not significant .
( c ) comparative quantification showing significant lower ratio of valves to lymphatic invasion area in response to anti 
the experiment was repeated twice with seven mice in control and eight mice in treatment group .
previously , we reported that corneal lymphatic vessels observe a unique nasal dominant distribution pattern in inflammatory lg . to investigate whether this phenomenon also manifests in transplantation - associated lg and whether it is affected by the itga-9 treatment , we next investigated the effect of itga-9 blockade on the polarity of lg by comparing the nasal and temporal quadrants , as illustrated in figure 2a .
our results showed that in both treatment and control groups , lymphatic vessels were more distributed at the nasal side , and itga-9 blockade had no effect on this polarity of corneal lg ( fig .
our further association analysis using the linear mixed model also confirmed that the polarized distribution of lg was associated only with corneal regions but not with the anti  itga-9 blockade .
integrin alpha 9 blockade had no effect on polarized distribution of lymphatic vessels after corneal transplantation .
( b ) comparative quantification showing significantly greater lymphatic invasion area in the nasal rather than the temporal side in both control and treatment conditions .
the experiment was repeated twice with seven mice in control and eight mice in treatment group .
* p < 0.05 . to further evaluate the effect of itga-9 blockade on corneal graft survival , we examined the grafts in both treatment and control groups and evaluated their survival rate twice a week up to 8 weeks after the surgery .
as shown in figure 3 , our results showed a significant promotion of graft survival by this treatment .
although graft rejection in both the control and treatment groups started approximately 2.5 weeks after transplantation , a significantly higher percentage of the grafts survived in the treatment group by the end of the 8-week study , as analyzed by the kaplan - meier survival curves ( p < 0.05 ) .
( a ) representative images from slit - lamp examination of rejected and survived grafts in control and treatment conditions , respectively .
( b ) kaplan - meier survival curves showing significantly higher survival rate in the treatment group . *
we first studied the effect of itga-9 blockade on corneal lg and vg induced by transplantation .
either itga-9 neutralizing body or isotype control was injected subconjunctivally twice a week starting from the surgery date . as demonstrated in figure 1a , following the treatment with the itga-9 blocking antibody , corneal lymphatic vessels contained significantly fewer valves compared with the control condition . summarized data from repetitive experiments are presented in figure 1b ( left ; p < 0.05 ) . however , this treatment had no effect on lg , as shown in figure 1b ( right ) .
our further analysis on the ratio of valve quantity to lymphatic invasion area revealed a significant reduction in this parameter in the treated rather than the control condition ( figure 1c ; p < 0.05 ) .
( a ) representative whole - mount immunostaining images demonstrating significantly fewer valves in the itga-9 blocking antibody  treated cornea in comparison with isotype control  treated cornea .
( b ) comparative quantification on lymphatic valves and lymphatic vessel invasion area in control and treatment conditions . anti 
the experiment was repeated twice with seven mice in control and eight mice in treatment group . * p < 0.05 ; n.s . , not significant .
( c ) comparative quantification showing significant lower ratio of valves to lymphatic invasion area in response to anti 
the experiment was repeated twice with seven mice in control and eight mice in treatment group .
previously , we reported that corneal lymphatic vessels observe a unique nasal dominant distribution pattern in inflammatory lg . to investigate whether this phenomenon also manifests in transplantation - associated lg and whether it is affected by the itga-9 treatment , we next investigated the effect of itga-9 blockade on the polarity of lg by comparing the nasal and temporal quadrants , as illustrated in figure 2a .
our results showed that in both treatment and control groups , lymphatic vessels were more distributed at the nasal side , and itga-9 blockade had no effect on this polarity of corneal lg ( fig .
our further association analysis using the linear mixed model also confirmed that the polarized distribution of lg was associated only with corneal regions but not with the anti  itga-9 blockade .
integrin alpha 9 blockade had no effect on polarized distribution of lymphatic vessels after corneal transplantation .
( b ) comparative quantification showing significantly greater lymphatic invasion area in the nasal rather than the temporal side in both control and treatment conditions .
the experiment was repeated twice with seven mice in control and eight mice in treatment group .
to further evaluate the effect of itga-9 blockade on corneal graft survival , we examined the grafts in both treatment and control groups and evaluated their survival rate twice a week up to 8 weeks after the surgery .
as shown in figure 3 , our results showed a significant promotion of graft survival by this treatment . although graft rejection in both the control and treatment groups started approximately 2.5 weeks after transplantation , a significantly higher percentage of the grafts survived in the treatment group by the end of the 8-week study , as analyzed by the kaplan - meier survival curves ( p < 0.05 ) .
( a ) representative images from slit - lamp examination of rejected and survived grafts in control and treatment conditions , respectively .
( b ) kaplan - meier survival curves showing significantly higher survival rate in the treatment group .
in this study , we demonstrated for the first time that itga-9 is critically involved in corneal transplantation  induced vg , and its molecular blockade can effectively suppress this process .
we have also shown that this treatment strategy does not affect corneal lg or its polarity of nasal distribution .
more importantly , we have offered the first evidence showing that by reducing the lymphatic valves but not the vessels themselves , we were able to achieve a higher rate of graft survival .
our finding that itga-9 blockade suppressed lymphatic valve formation without disturbing the lymphatic vessels in transplantation is consistent with our previous report on a suture - induced inflammation model .
this may be explained by the fact that itga-9 is more highly expressed on the valves than the vessel walls , as shown in supplementary figure 2 with a transplanted cornea .
the disparity between lymphatic valves and vessels was also observed during development , in which a reduced number of lymphatic valves , but not vessels , were detected in itga-9 knockout mice . with the treatment regimen used in this study
, we did not observe any obvious side effects . for future development of clinical application , it may be possible to achieve the therapeutic effects by using various formats of the antagonists against the itga-9 pathway , such as neutralizing antibodies or small molecules , which merits further investigation and is beyond the scope of this report .
this finding indicates a compromise of the immune reflex arc in which the lymphatic pathway serves as the afferent arm .
it also aligns well with a previous developmental report that itga-9 knockout mice died shortly after birth from bilateral chylothorax , in which lymphatic vessels were present but displayed compromised integrity .
moreover , we have reported that lymphatic vessels are equipped with valves as they become mature and functional .
therefore , by targeting on lymphatic valves , we may have interfered with the maturation process of the lymphatic vessels , rendering them dysfunctional .
it would be interesting to check if this strategy also affects other indices of the immune responses involved in transplant rejection , such as delayed - type hypersensitivity , which warrants further investigation . in summary
it also provides a novel therapeutic strategy to effectively interfere with this pathological process and to improve graft survival .
as one of the favorite tools for lymphatic study in general , our results from the cornea may shed some light on the development of new itga-9based therapies to treat broader lymphatic and immune diseases in the body .
 purposethe lymphatic pathway mediates transplant rejection . 
 we recently reported that lymphatic vessels develop luminal valves in the cornea during lymphangiogenesis , and these valves express integrin alpha 9 ( itga-9 ) and play a critical role in directing lymph flow . in this study , we used an allogeneic corneal transplantation model to investigate whether itga-9 blockade could suppress valvulogenesis after transplantation , and how this effect would influence the outcomes of the transplants.methodsorthotopic corneal transplantation was performed between fully mismatched c57bl/6 ( donor ) and balb / c ( recipient ) mice . 
 the recipients were randomized to receive subconjunctival injections of either itga-9 blocking antibody or isotype control twice a week for 8 weeks . 
 corneal grafts were assessed in vivo by ophthalmic slit - lamp biomicroscopy and analyzed using kaplan - meier survival curves . additionally , whole - mount full - thickness corneas were evaluated ex vivo by immunofluorescent microscopy on both lymphatic vessels and valves.resultsantiitga-9 treatment suppressed lymphatic valvulogenesis after transplantation . 
 our treatment did not affect lymphatic vessel formation or their nasal polarized distribution in the cornea . 
 more importantly , itga-9 blockade led to a significant promotion of graft survival.conclusionslymphatic valvulogenesis is critically involved in transplant rejection . 
 itga-9 targeting may offer a new and effective strategy to interfere with the immune responses and promote graft survival . 
Methods
Animals
Corneal Transplantation
Pharmaceutical Intervention
In Vivo Assessment of Grafted Corneas
Corneal Immunofluorescent Microscopy
Lymphatic Vessel and Valve Quantification
Statistical Analysis
Results
Effect of Itga-9 Blockade on Lymphatic Valvulogenesis After Corneal Transplantation
Effect of Itga-9 Blockade on Nasal Dominant Distribution of Lymphatic Vessels
Effect of Itga-9 Blockade on Corneal Graft Survival
Discussion
Supplementary Material