AB027.Varying pattern of proteases secretion in Fuchs corneal endothelial dystrophy

Background:The goal of this project was to analyze the relationship between cell morphology and proteases/proteases inhibitors(PIs)secretion profile in fuchs endothelial corneal dystrophy(FECD)corneal endothelial cells(CECs).Methods:Cell morphology was determined using a circularity index(4π×area/perimeter2)for each CECs population extracted from surgical FECD specimens(N=2)and healthy Eye bank corneas(N=3).CECs were cultured 28 days post-confluency.Supernatant was collected and analysed using Proteome Profiler Array detecting 35 proteases and 32 PIs(R&D Systems).Proteome signal was analyzed using Image Studio Lite and correlated with the population’s circularity index.Results:Calculation of circularity index reported different morphologies among FECD populations(0.59±0.18 and 0.64±0.17)and healthy populations(0.44±0.18,0.66±0.13 and 0.71±0.11).Proteome arrays revealed the presence of 10 proteases(ADAMTS1,Cathepsin A,B,D,and X/Z/P,DPPIV/CD26,MMP-2,3 and 12,uPA/Urokinase)and 10 PIs(Protease Nexin II,Cystatin B and C,EMMPRIN/CD147,Latexin,Lipocalin-1,Serpin E1,TFPI,TFPI-2,TIMP-1,2 and 4).Healthy and FECD specimens showed similar variation patterns according to morphology for secretion of ADAMTS1,MMP-3 and 12.However,opposing patterns between healthy and FECD populations were observed for Cathepsin B and D.Moreover,some proteins did not show variation according to phenotype in healthy CECs,but did in FECD CECs:Cathepsin A,Cystatin C,TFPI-2 and total TIMPs.For the other proteins,secretion did not vary according to morphology or no specific pattern was distinguishable.Conclusions:To conclude,our results suggest that cell phenotype is linked to the secretion of certain proteases/PIs in both groups.However,there seems to be differences in secretion of particular proteases and PIs between FECD and healthy specimens as morphology did not have a similar influence.These differences might initiate an imbalance between proteases and PIs explaining the irregular thickening of the Descemet membrane seen in FECD.

AB089.Impact of WNK1 inhibition on corneal wound healing using a model of human tissue-engineered cornea

Background:Because of its superficial anatomical localization,the cornea is particularly vulnerable to abrasive forces and various traumas,which can lead to significant visual impairments.Upon injury of the corneal epithelium,there are important changes that occur in the composition of the underlying extracellular matrix(ECM).Those changes are perceived by the integrins that recognize the ECM components as their ligand and activate different intracellular signalling pathways,ultimately leading to reepithelialisation and reorganization of the injured epithelium,both of which are necessary in order to restore the visual properties of the cornea.The goal of this study was to analyse the impact of the pharmacological inhibition of specific signal transduction mediators of integrin-dependant signalling pathways on corneal wound healing using both monolayers of hCECs and tissue-engineered human corneas(hTECs)as in vitro models.Methods:hTECs were produced by the self-assembly approach and wounded with a 8-mm diameter biopsy punch.Total RNA and proteins were isolated from the wounded and unwounded hTECs to conduct gene profiling analyses and protein kinase arrays.The wounded tissues were then incubated with the WNK1 inhibitor WNK463 and wound healing was monitored over a period of 6 days.Control corneas were incubated with the vehicle alone(DMSO).The impact of WNK1 inhibition on hCECs monolayers was determined using a scratch wound assay.Results:Gene profiling analyses and protein kinases arrays revealed important alterations in the expression and activity of several mediators from the integrin-dependent signalling pathways in response to the ECM changes taking place during corneal wound healing.Among these,WNK1 is considerably activated through phosphorylation during corneal wound healing.The pharmacological inhibition of WNK1 by WNK463 significantly reduced the dynamic of corneal wound closure in our hTECs and hCECs monolayers compared to their respective negative controls.Conclusions:These results allowed the identification of WNK1 kinase as an important player for a proper healing of the cornea.Also,these results allowed for a better understanding of the cellular and molecular mechanisms involved in corneal wound healing and they may lead to the identification of new therapeutic targets in the field of corneal wounds.

AB047.Quebec database of clinical data and biological material for research on uveal melanoma

Background:Sharing biological material and clinical data from patients with uveal melanoma.Methods:Uveal melanoma is the most common intraocular malignancy in the adult population.Because uveal melanoma is primarily a sporadic cancer and familial cases are rare,it is difficult to prevent or detect it.Despite effective treatment of ocular tumors,more than 50%of patients develop incurable liver metastases mainly in the 5-10 years following the detection of the primary tumor.This cancer is relatively rare and the obtained biopsies are very small.About 20 samples are taken each year in Quebec.This provincial infrastructure is made of biological material from donors with uveal melanoma and a large clinical database.Collected tumor biopsies are used for culturing cell lines and the creation of a DNA/RNA library used for genomic and genetic studies.Results:This infrastructure plays an important role in the achievement of various research programs for a better understanding of genetic and environmental factors involved in the development of melanoma and the spread of metastasis.It allows collaboration with other researchers at a provincial,national and international level in order to make progress in basic and clinical research on uveal melanoma.Conclusions:The biological material and clinical data of this infrastructure are available upon request to VHRN members whose research project was approved by the ethics committee of the institution.

AB021.Discovery of a new role for the WNK1 kinase in corneal wound healing

Background:Damage to the corneal epithelium triggers important changes in the composition of the extracellular matrix(ECM)to which the basal human corneal epithelial cells(hCECs)attach.These changes are perceived by integrins,a family of trans-membrane receptors that activate different intracellular signalling pathways,ultimately leading to re-epithelialization of the injured epithelium.Our goal is to study the impact of the pharmacological inhibition/activation of specific signal transduction mediators on corneal wound healing using both monolayers of hCECs and tissue-engineered human corneas(hTECs)as in vitro models.Methods:hTECs were produced by the self-assembly approach and wounded with a 8-mm biopsy punch.Total RNA and proteins were isolated from the wounded and unwounded hTECs to conduct gene profiling analyses and protein kinase arrays.The wounded tissues were then incubated either with the lysine deficient protein kinase 1(WNK1)inhibitor WNK463,the WNK1 indirect agonist AM1241,or with the vehicle alone(DMSO;negative control)and wound healing was monitored for 6 days.The impact of WNK1 inhibition/activation on hCECs monolayers was determined using scratch wound assays.Results:Gene profiling analyses and protein kinases arrays revealed that expression and activity of several mediators from the integrin-dependent signalling pathways were altered in response to the ECM changes taking place during corneal wound healing.Phosphorylation of the WNK1 kinase turned out to be the most striking activation event occurring during wound healing.Since the pharmacological inhibition of WNK1 by WNK463 significantly reduced the rate of corneal wound closure in our hTECs and hCECs monolayers compared to their respective negative controls,we believe that the pharmacological activation of WNK1 could turn out to be an interesting avenue to accelerate corneal wound closure.Conclusions:These results will contribute to a better understanding of the cellular and molecular mechanisms involved in corneal wound healing.Furthermore,they identified a new function for the WNK1 kinase in corneal wound healing and might lead to the identification of a new therapeutic target in the field of corneal wounds.

AB030. Characterization of the interactions between hepatic stellate cells and tumor cells during uveal melanoma metastatic progression

Background:Uveal melanoma(UM)is the most common primary eye tumor in adults,and the most frequent site of malignant transformation of the melanocytes after the skin.It spreads to the liver in half of the cases,and the death rate following the report of metastasis is 92%at 2 years.Hepatic tropism of UM cannot simply be explained by the blood circulatory system organization,and illustrates the“seed and soil”hypothesis that describes an interaction between tumor cells(seed)and a specific microenvironment(soil).We decided to focus our study on the synergic interaction between UM cells and hepatic stellate cells(HSC),whose role has been previously described in the metastatic progression of colon and pancreatic cancers.Furthermore,HSC have been found surrounding UM liver metastasis,and the UM secretome contains activating cytokines of hepatic stromal cells.Our hypothesis is that HSC provide a specific microenvironment in the liver enhancing the growth of UM cells and increasing their therapeutic resistance.Using an in vitro 3D model and an original xenograft mouse model,we aim to decipher the mechanisms of UM metastatic progression,in order to elaborate new therapeutic strategies.Methods:First,using an agar coating,spheroids were generated with UM cells and were allowed to grow for 72 h.These tumor spheroids were then embedded in Matrigel and the HSC conditioned medium was used to evaluate the impact of the HSC secretome on UM invasion.Next,an original in vivo xenograft mouse model was generated,in which metastatic UM cells were injected alone or with human HSC in the spleen of immunodeficient mice.This model allows us to evaluate in 3-6 weeks the metastatic potential of each cell population,and thus to determine the cooperation between HSC and UM cells in the liver.Results:The HSC conditioned medium increased the invasion of UM spheroids compared to non-conditioned medium in our in vitro model.In addition,UM cells inoculated in the mouse spleen alone or with human HSC were able to metastasize to the liver,and the host HSC were also recruited by UM metastases.Conclusions:Our preliminary results strongly suggest that the secretome of HSC provides a permissive microenvironment for UM metastatic progression.We now have to confirm these results by characterizing the secretome of HSC,in order to identify cytokines or growth factors that increase the invasion of the liver by UM.Our models can be used to test the efficacy of new therapeutic strategies targeting the UM microenvironment.

AB023.Ultrastable gold nanoparticles as a drug delivery system for ocular therapy

Background:Ocular therapy administrated by ophthalmic drops is advantageous thanks to its simplicity.However,efficiency of active molecules is limited when administered by this method.Indeed,more than 99.9%is discarded due to multiple factors including lacrimal drainage.Low retention time of drugs at the cornea leads to their poor penetration.Our hypothesis is that a drug delivery system based on gold nanoparticles should enhance the efficiency of the drugs.The main objective is to develop new methods to improve active molecules biodisponibility in ocular therapy thanks to a new drug delivery system implying gold nanoparticles.The specific objectives are:(I)to synthesize and characterize ultrastable gold nanoparticles,(II)to establish the drug encapsulation protocol,(III)to develop a separation method of free and encapsulated drugs to allow their quantification,(IV)to study the cytotoxicity of our gold nanoparticles.Methods:Ultrastable gold nanoparticles were synthesized by a new method and their ultrastability toward several harsh conditions was characterized.An encapsulation protocol was settled for several drugs.The separation of free and encapsulated drugs was performed with magnetic beads.The quantification of the encapsulated drugs was performed by HPLC.A MTS assay was performed on 3 corneal epithelial cell populations,exposed or unexposed to gold nanoparticles.Reconstructed corneas were prepared using the self-assembly method.A wound healing experience was performed on those corneas with or without nanoparticles.Results:Gold nanoparticles were synthesized and purified according to our new experimental conditions.They support harsh conditions as several cycles of freeze-drying,heating,salt exposition and ultracentrifugation.For the first time in literature,gold nanoparticle support autoclave sterilisation.The separation method involving magnetic beads was optimized to get rid of non-specific interactions.The encapsulation efficiency varies according to the active molecule.The MTS assay did not show diminution of the cellular viability when in presence of gold nanoparticles.Furthermore,gold nanoparticle exposition did not slow the wound healing of reconstructed corneas.Conclusions:Our new ultrastable gold nanoparticles can have a major impact in nanomedicine.They can support harsh conditions,as autoclave treatment,allowing their sterilisation for in vivo use.We showed that active molecules can be encapsulated in gold nanoparticles.In addition,they do not seem to cause any diminution of cellular viability.These data suggest the possible improvements in ocular therapy thanks to gold nanoparticles.