Background:Our previous studies revealed that Nogo-A gene ablation improved visual function recovery after retinal injury.Moreover,Nogo-A expression is highly expressed in the healthy retina.Its physiological role in ...Background:Our previous studies revealed that Nogo-A gene ablation improved visual function recovery after retinal injury.Moreover,Nogo-A expression is highly expressed in the healthy retina.Its physiological role in retinal function is not known.The purpose of this current study was to determine the effects of acute Nogo-A silencing on retinal neuron structure and function in physiological conditions.Methods:Nogo-A silencing was done by intravitreal injection of adeno-associated virus serotype 2.2 containing a short hairpin RNA sequence(AAV2.2 shRNA-Nogo-A)and a GFP reporter gene in adult C57BL/6J mice.As control,an empty AAV2.2 vector was used.Infection of retinal cells was followed by fluorescent fundoscopy.Changes in Nogo-A expression were analysed by Western blotting in whole retinal lysates.Electroretinography was used to monitor retinal activity.The assessment of optokinetic reflex(OKR)allowed to follow visual acuity in unrestrained mice.Immunofluorescence on histological sections using the following cell markers,i.e.,RNA-binding protein with multiple splicing(RBPMS)and sex-determining region Y-box 2(Sox-2)allowed to visualize retinal ganglion cells(RGCs)and Müller glia respectively.Results:GFP fluorescence revealed efficient AAV2.2 transfection in the ganglion cell layer and the inner nuclear layer 30 days after viral injection.By Western blotting,Nogo-A expression was decreased by~75%in AAV2.2-shRNA-Nogo-A-treated retinae(n=3)as compared to the control mice(n=3).Strikingly,AAV2.2-shRNA-Nogo-A-injected animals(n=10)had a visual acuity reduction of 43.7%as compared to control(n=7),60 days after transfection.Electroretinography(ERG)b-wave and a-wave amplitudes were also decreased by~35%and 24.4%respectively relative to controls.After two months of transfection,RBPMS-positive RGCs were reduced by~30%in AAV2.2-shRNA-Nogo-A(n=4)compared to non-injected contralateral eyes(n=4).The number of Sox2-expressing Müller cells was not affected after Nogo-A knockdown.Conclusions:Nogo-A gene silencing in the retina has deleterious effects on the mouse retinal structure and function,suggesting an important role for Nogo-A in retinal physiology.展开更多
Background:In glaucoma and after an ischemic injury of the retina,excessive activation of N-Methyl-D-Aspartate receptors,a type of glutamatergic receptors,induces the death of retinal ganglion cells and an irreversibl...Background:In glaucoma and after an ischemic injury of the retina,excessive activation of N-Methyl-D-Aspartate receptors,a type of glutamatergic receptors,induces the death of retinal ganglion cells and an irreversible vision loss.The painless loss of retinal cells does not allow for a swift diagnostic and treatment of retinal damages.There is no efficient therapy to improve retinal functions in this case.In order to develop new therapeutic approaches for retinal injury,we propose,in this study,to stimulate neuronal plasticity of the visual system by neutralising the glial protein,Nogo-A.The inhibitory action of this protein on axonal regeneration is well known in spinal cord injuries but not in the visual system.We thus studied the function of Nogo-A in vision recovery in mice.Methods:Nogo-A activities were chronically blocked by deleting its gene in KO mice and acutely,by intravitreal injections of an antibody known as 11C7.Inner retina lesions were done by injection of 0.5 or 5 nmol of NMDA in the vitreous humor.A PBS buffer was administered in control animals.The visual system functions were accessed with an optokinetic test in awake mice,by electroretinography(ERG)in the eye and visual evoked potentials in the visual cortex.Cell survival of retinal ganglion cells,amacrine and bipolar cells was evaluated on histological sections by immunofluorescence.Changes in expression of Nogo-A,its receptors and neuronal plasticity associated molecules were observed by Western blot and q-PCR.Results:At NMDA doses of≤0.5 nmol,Nogo-A KO mice show a recovery of optokinetic responses much faster than in WT mice.Surprisingly,a single injection of the antibody,11C7 was sufficient to improve VEPs of NMDA injured animals as compared to control antibody.Furthermore,ERGs showed that a dose of 0.5 nmol induced retinal lesions limited to the ganglion cell layer,with significant changes to the VEPs but without influencing photoreceptors and inner nuclear layer cells functions.However,5 nmol of NMDA affected the survival of inner nuclear layer cells and reduced by~50%their activity.Conclusions:Our results show that the neutralisation of Nogo-A can improve visual functions after injury to the inner retina.Inhibition of Nogo-A could thus be an efficient way to treat pathologies like glaucoma.展开更多
Background:Glaucoma is an optical neuropathy affecting over 67 million people in the world.Efficiency of current active molecules,as travoprost(hydrophobic)is limited when administered by ophthalmic drops.Indeed,more ...Background:Glaucoma is an optical neuropathy affecting over 67 million people in the world.Efficiency of current active molecules,as travoprost(hydrophobic)is limited when administered by ophthalmic drops.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.The aim of the project is to develop a drug delivery system allowing the drug penetration through biological barriers.Our hypothesis is that a drug delivery system based on gold nanoparticles should enhance the efficiency of the drugs.The main objective is to study the encapsulation ability of gold nanoparticles towards travoprost.The specific objectives are(I)the synthesis and characterizations of gold nanoparticles;(II)the establishment of the encapsulation protocol;(III)the method development of the separation of free and encapsulated drugs and;(IV)the quantification of the encapsulated drugs.Methods:Gold nanoparticles were synthesized by a new method developed in our laboratory.An encapsulation protocol was settled using aqueous conditions at 37℃.The separation of free and encapsulated drugs was performed with magnetic beads.The quantification of the encapsulated drugs was then performed by high performance liquid chromatography and confirmed by UV-visible spectroscopy.Results:Gold nanoparticles of 28±1 nm were synthesized and purified according to our new experimental conditions.The encapsulation protocol lasts 5 days in the optimised conditions.The separation method involving magnetic beads was optimized to get rid of non-specific interactions.The travoprost was incubated with the nanoparticles until the reach of equilibrium in solution.Conclusions:We showed that active molecules used for glaucoma therapy,as travoprost,can be encapsulated in gold nanoparticles.Further analysis will allow identifying the encapsulation properties of various gold nanoparticles,differing by their size,shape and chemical surface.These data suggest the possible improvements.展开更多
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 discarde...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.展开更多
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 ep...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.展开更多
Background:Retinol dehydrogenase 8(RDH8)is a 312-amino acid(aa)protein involved in the visual cycle.Bound to the outer segment disk membranes of photoreceptors,it reduces all-trans-retinal to all-trans-retinol1 as one...Background:Retinol dehydrogenase 8(RDH8)is a 312-amino acid(aa)protein involved in the visual cycle.Bound to the outer segment disk membranes of photoreceptors,it reduces all-trans-retinal to all-trans-retinol1 as one of the rate-limiting steps of the visual cycle2.RDH8 is a member of the short-chain dehydrogenase/reductase family.Its C-terminal segment allows its membrane-anchoring through the postulated presence of an amphipathicα-helix and of 1 to 3 acyl groups at positions 299,302 and 3043.The secondary structure and membrane binding characteristics of RDH8 and its C-terminal segment have not yet been described.Methods:To evaluate the membrane binding of RDH8,the full-length protein(aa 1-312),a truncated form(aa 1-296),its C-terminal segment(aa 281-312 and 297-312)as well as different additional variants of this segment were used.The truncated protein binds membranes less efficiently than the full-length form.Thus,the C-terminal segment of RDH8 is essential for the binding and has thus been further examined.The intrinsic fluorescence of tryptophan residues at positions 289 and 310 of the wild-type C-terminal segment of RDH8 and the mutants W289F,W310F and W310R have thus been used to determine their extent of binding to lipid vesicles and to monitor their local environment.Unilamellar lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)or a mixture of POPC and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine(POPS)were used to mimic the phospholipid content of the outer segment disk membranes of photoreceptors.Results:An increase in fluorescence intensity and in fluorescence lifetime is observed upon increasing the concentration of lipid vesicles.These data allowed calculating values of partition coefficient of the C-terminal segment of RDH8 varying between Kp=1.1 E6 to 1.7 E6.It is noteworthy that the observation of a more intense shift to lower wavelengths upon membrane binding of the mutant W310R and W310F indicates a deeper incorporation of the remaining tryptophan residue at position 289 into the lipid bilayer.The secondary structure of the C-terminal segment of RDH8 observed by circular dichroism and infrared spectroscopy shows a superposition ofα-helical,β-turn and unordered structures.Conclusions:The peptides derived from the C-terminal segment of RDH8 show a strong binding to lipid vesicles.These strength of binding is independent of the type of lipid and the presence of a mutation.展开更多
Background:The cornea composes the outer surface of the eye and its transparency is required to allow light transmission to the retina.However,because of its position,the cornea is subjected to chemical and mechanical...Background:The cornea composes the outer surface of the eye and its transparency is required to allow light transmission to the retina.However,because of its position,the cornea is subjected to chemical and mechanical injuries that may lead to blindness.Our studies conducted using the human tissue-engineered cornea(hTEC)as a model provided evidence that the cyclic-AMP-response element binding protein(CREB)pathway is repressed during closure of corneal wounds.Based on these results,we hypothesized that closure of corneal wounds can be enhanced by preventing activation of CREB with the pharmacological inhibitor C646.Our goals were to proceed to the pharmacological inhibition of CREB(I)in vitro using the hTECs as a model,and then(II)in vivo using the rabbit as a model.Methods:The self-assembly approach was used to create hTECs,that were then wounded with an 8-mm diameter biopsy punch to create an epithelial defect.The tissues were then incubated with 10μM of C646(n=8).DMSO was used alone as a negative control(n=4).Closure of the wounds was monitored over a period of 5 days.Besides,the cornea of New Zealand white rabbits was debrided with an ethanol 70%solution to create an epithelial defect of 8-mm diameter.Several concentrations of C646(1,10,100μM et 1 mM)were applied as eye drops 3 times a day for up to 7 days.The wounded corneas(n=4 per concentration)were stained with fluorescein and photographed every day.Results:In vitro pharmacological inhibition of CREB with C646 considerably accelerated wound closure of all treated hTECs(4 days)compared to the control group(7 days).Moreover,the in vivo C646 treatment also accelerated wound healing of the corneas compared to the control group.The most effective concentration of C646 tested was the lowest(1μM),as it considerably enhanced the wound healing process.Conclusions:This study demonstrates that wound healing both in vitro and in vivo can be enhanced by preventing activation of CREB using a pharmacological inhibition approach.Most of all,this experiment suggests mediators from the CREB pathway as potential therapeutic targets on which we may influence to alter the wound healing dynamic of the cornea.We believe this study will lead to significant advancements in the clinical field of corneal defects.展开更多
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 i...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.展开更多
文摘Background:Our previous studies revealed that Nogo-A gene ablation improved visual function recovery after retinal injury.Moreover,Nogo-A expression is highly expressed in the healthy retina.Its physiological role in retinal function is not known.The purpose of this current study was to determine the effects of acute Nogo-A silencing on retinal neuron structure and function in physiological conditions.Methods:Nogo-A silencing was done by intravitreal injection of adeno-associated virus serotype 2.2 containing a short hairpin RNA sequence(AAV2.2 shRNA-Nogo-A)and a GFP reporter gene in adult C57BL/6J mice.As control,an empty AAV2.2 vector was used.Infection of retinal cells was followed by fluorescent fundoscopy.Changes in Nogo-A expression were analysed by Western blotting in whole retinal lysates.Electroretinography was used to monitor retinal activity.The assessment of optokinetic reflex(OKR)allowed to follow visual acuity in unrestrained mice.Immunofluorescence on histological sections using the following cell markers,i.e.,RNA-binding protein with multiple splicing(RBPMS)and sex-determining region Y-box 2(Sox-2)allowed to visualize retinal ganglion cells(RGCs)and Müller glia respectively.Results:GFP fluorescence revealed efficient AAV2.2 transfection in the ganglion cell layer and the inner nuclear layer 30 days after viral injection.By Western blotting,Nogo-A expression was decreased by~75%in AAV2.2-shRNA-Nogo-A-treated retinae(n=3)as compared to the control mice(n=3).Strikingly,AAV2.2-shRNA-Nogo-A-injected animals(n=10)had a visual acuity reduction of 43.7%as compared to control(n=7),60 days after transfection.Electroretinography(ERG)b-wave and a-wave amplitudes were also decreased by~35%and 24.4%respectively relative to controls.After two months of transfection,RBPMS-positive RGCs were reduced by~30%in AAV2.2-shRNA-Nogo-A(n=4)compared to non-injected contralateral eyes(n=4).The number of Sox2-expressing Müller cells was not affected after Nogo-A knockdown.Conclusions:Nogo-A gene silencing in the retina has deleterious effects on the mouse retinal structure and function,suggesting an important role for Nogo-A in retinal physiology.
文摘Background:In glaucoma and after an ischemic injury of the retina,excessive activation of N-Methyl-D-Aspartate receptors,a type of glutamatergic receptors,induces the death of retinal ganglion cells and an irreversible vision loss.The painless loss of retinal cells does not allow for a swift diagnostic and treatment of retinal damages.There is no efficient therapy to improve retinal functions in this case.In order to develop new therapeutic approaches for retinal injury,we propose,in this study,to stimulate neuronal plasticity of the visual system by neutralising the glial protein,Nogo-A.The inhibitory action of this protein on axonal regeneration is well known in spinal cord injuries but not in the visual system.We thus studied the function of Nogo-A in vision recovery in mice.Methods:Nogo-A activities were chronically blocked by deleting its gene in KO mice and acutely,by intravitreal injections of an antibody known as 11C7.Inner retina lesions were done by injection of 0.5 or 5 nmol of NMDA in the vitreous humor.A PBS buffer was administered in control animals.The visual system functions were accessed with an optokinetic test in awake mice,by electroretinography(ERG)in the eye and visual evoked potentials in the visual cortex.Cell survival of retinal ganglion cells,amacrine and bipolar cells was evaluated on histological sections by immunofluorescence.Changes in expression of Nogo-A,its receptors and neuronal plasticity associated molecules were observed by Western blot and q-PCR.Results:At NMDA doses of≤0.5 nmol,Nogo-A KO mice show a recovery of optokinetic responses much faster than in WT mice.Surprisingly,a single injection of the antibody,11C7 was sufficient to improve VEPs of NMDA injured animals as compared to control antibody.Furthermore,ERGs showed that a dose of 0.5 nmol induced retinal lesions limited to the ganglion cell layer,with significant changes to the VEPs but without influencing photoreceptors and inner nuclear layer cells functions.However,5 nmol of NMDA affected the survival of inner nuclear layer cells and reduced by~50%their activity.Conclusions:Our results show that the neutralisation of Nogo-A can improve visual functions after injury to the inner retina.Inhibition of Nogo-A could thus be an efficient way to treat pathologies like glaucoma.
文摘Background:Glaucoma is an optical neuropathy affecting over 67 million people in the world.Efficiency of current active molecules,as travoprost(hydrophobic)is limited when administered by ophthalmic drops.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.The aim of the project is to develop a drug delivery system allowing the drug penetration through biological barriers.Our hypothesis is that a drug delivery system based on gold nanoparticles should enhance the efficiency of the drugs.The main objective is to study the encapsulation ability of gold nanoparticles towards travoprost.The specific objectives are(I)the synthesis and characterizations of gold nanoparticles;(II)the establishment of the encapsulation protocol;(III)the method development of the separation of free and encapsulated drugs and;(IV)the quantification of the encapsulated drugs.Methods:Gold nanoparticles were synthesized by a new method developed in our laboratory.An encapsulation protocol was settled using aqueous conditions at 37℃.The separation of free and encapsulated drugs was performed with magnetic beads.The quantification of the encapsulated drugs was then performed by high performance liquid chromatography and confirmed by UV-visible spectroscopy.Results:Gold nanoparticles of 28±1 nm were synthesized and purified according to our new experimental conditions.The encapsulation protocol lasts 5 days in the optimised conditions.The separation method involving magnetic beads was optimized to get rid of non-specific interactions.The travoprost was incubated with the nanoparticles until the reach of equilibrium in solution.Conclusions:We showed that active molecules used for glaucoma therapy,as travoprost,can be encapsulated in gold nanoparticles.Further analysis will allow identifying the encapsulation properties of various gold nanoparticles,differing by their size,shape and chemical surface.These data suggest the possible improvements.
文摘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.
文摘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.
文摘Background:Retinol dehydrogenase 8(RDH8)is a 312-amino acid(aa)protein involved in the visual cycle.Bound to the outer segment disk membranes of photoreceptors,it reduces all-trans-retinal to all-trans-retinol1 as one of the rate-limiting steps of the visual cycle2.RDH8 is a member of the short-chain dehydrogenase/reductase family.Its C-terminal segment allows its membrane-anchoring through the postulated presence of an amphipathicα-helix and of 1 to 3 acyl groups at positions 299,302 and 3043.The secondary structure and membrane binding characteristics of RDH8 and its C-terminal segment have not yet been described.Methods:To evaluate the membrane binding of RDH8,the full-length protein(aa 1-312),a truncated form(aa 1-296),its C-terminal segment(aa 281-312 and 297-312)as well as different additional variants of this segment were used.The truncated protein binds membranes less efficiently than the full-length form.Thus,the C-terminal segment of RDH8 is essential for the binding and has thus been further examined.The intrinsic fluorescence of tryptophan residues at positions 289 and 310 of the wild-type C-terminal segment of RDH8 and the mutants W289F,W310F and W310R have thus been used to determine their extent of binding to lipid vesicles and to monitor their local environment.Unilamellar lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)or a mixture of POPC and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine(POPS)were used to mimic the phospholipid content of the outer segment disk membranes of photoreceptors.Results:An increase in fluorescence intensity and in fluorescence lifetime is observed upon increasing the concentration of lipid vesicles.These data allowed calculating values of partition coefficient of the C-terminal segment of RDH8 varying between Kp=1.1 E6 to 1.7 E6.It is noteworthy that the observation of a more intense shift to lower wavelengths upon membrane binding of the mutant W310R and W310F indicates a deeper incorporation of the remaining tryptophan residue at position 289 into the lipid bilayer.The secondary structure of the C-terminal segment of RDH8 observed by circular dichroism and infrared spectroscopy shows a superposition ofα-helical,β-turn and unordered structures.Conclusions:The peptides derived from the C-terminal segment of RDH8 show a strong binding to lipid vesicles.These strength of binding is independent of the type of lipid and the presence of a mutation.
文摘Background:The cornea composes the outer surface of the eye and its transparency is required to allow light transmission to the retina.However,because of its position,the cornea is subjected to chemical and mechanical injuries that may lead to blindness.Our studies conducted using the human tissue-engineered cornea(hTEC)as a model provided evidence that the cyclic-AMP-response element binding protein(CREB)pathway is repressed during closure of corneal wounds.Based on these results,we hypothesized that closure of corneal wounds can be enhanced by preventing activation of CREB with the pharmacological inhibitor C646.Our goals were to proceed to the pharmacological inhibition of CREB(I)in vitro using the hTECs as a model,and then(II)in vivo using the rabbit as a model.Methods:The self-assembly approach was used to create hTECs,that were then wounded with an 8-mm diameter biopsy punch to create an epithelial defect.The tissues were then incubated with 10μM of C646(n=8).DMSO was used alone as a negative control(n=4).Closure of the wounds was monitored over a period of 5 days.Besides,the cornea of New Zealand white rabbits was debrided with an ethanol 70%solution to create an epithelial defect of 8-mm diameter.Several concentrations of C646(1,10,100μM et 1 mM)were applied as eye drops 3 times a day for up to 7 days.The wounded corneas(n=4 per concentration)were stained with fluorescein and photographed every day.Results:In vitro pharmacological inhibition of CREB with C646 considerably accelerated wound closure of all treated hTECs(4 days)compared to the control group(7 days).Moreover,the in vivo C646 treatment also accelerated wound healing of the corneas compared to the control group.The most effective concentration of C646 tested was the lowest(1μM),as it considerably enhanced the wound healing process.Conclusions:This study demonstrates that wound healing both in vitro and in vivo can be enhanced by preventing activation of CREB using a pharmacological inhibition approach.Most of all,this experiment suggests mediators from the CREB pathway as potential therapeutic targets on which we may influence to alter the wound healing dynamic of the cornea.We believe this study will lead to significant advancements in the clinical field of corneal defects.
文摘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.
