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Clusterin Seals the Ocular Surface Barrier in Mouse Dry Eye.

Bauskar A, Mack WJ, Mauris J, Argüeso P, Heur M, Nagel BA, Kolar GR, Gleave ME, Nakamura T, Kinoshita S, Moradian-Oldak J, Panjwani N, Pflugfelder SC, Wilson MR, Fini ME, Jeong S - PLoS ONE (2015)

Bottom Line: When the CLU level drops below the critical all-or-none threshold, the barrier becomes vulnerable to desiccating stress.CLU binds selectively to the ocular surface subjected to desiccating stress in vivo, and in vitro to the galectin LGALS3, a key barrier component.Positioned in this way, CLU not only physically seals the ocular surface barrier, but it also protects the barrier cells and prevents further damage to barrier structure.

View Article: PubMed Central - PubMed

Affiliation: USC Institute for Genetic Medicine and Graduate Program in Medical Biology, Keck School of Medicine of USC, University of Southern California, Los Angeles, California, United States of America.

ABSTRACT
Dry eye is a common disorder caused by inadequate hydration of the ocular surface that results in disruption of barrier function. The homeostatic protein clusterin (CLU) is prominent at fluid-tissue interfaces throughout the body. CLU levels are reduced at the ocular surface in human inflammatory disorders that manifest as severe dry eye, as well as in a preclinical mouse model for desiccating stress that mimics dry eye. Using this mouse model, we show here that CLU prevents and ameliorates ocular surface barrier disruption by a remarkable sealing mechanism dependent on attainment of a critical all-or-none concentration. When the CLU level drops below the critical all-or-none threshold, the barrier becomes vulnerable to desiccating stress. CLU binds selectively to the ocular surface subjected to desiccating stress in vivo, and in vitro to the galectin LGALS3, a key barrier component. Positioned in this way, CLU not only physically seals the ocular surface barrier, but it also protects the barrier cells and prevents further damage to barrier structure. These findings define a fundamentally new mechanism for ocular surface protection and suggest CLU as a biotherapeutic for dry eye.

No MeSH data available.


Related in: MedlinePlus

Topical CLU directly seals the ocular surface barrier disrupted by desiccating stress.The standard desiccating stress (DS) protocol was applied for 5-days to create ocular surface disruption. Non-stressed (NS) mice housed under normal ambient conditions served as a baseline control. Eyes with desiccating stress were then treated topically, a single time, with 1 uL of CLU formulated in PBS, 1 uL of BSA formulated in PBS for comparison, or 1 uL of PBS control. Barrier disruption was assayed by measuring corneal epithelial uptake of fluorescein (FU = Fluorescence Units at 521 nm). Values are expressed as the mean ± SD. (A) Eyes were treated a single time with recombinant human CLU (rhCLU) at 1, 3, 6 or 10 ug/mL, BSA at 10 ug/mL, or PBS. Fifteen minutes later, the fluorescein uptake test was performed, before there was time for barrier repair to occur. *P<0.0001 (n = 4). (B) Images of central cornea from the experiment shown in (A), obtained using laser scanning confocal microscopy at 10X magnification. One representative image out of two independent experiments is shown. Scale bar = 100 um. (C) Eyes were treated a single time with recombinant human CLU (rhCLU) at 10 ug/mL (right eyes) or PBS (left eyes). Then the mice were kept further for 2 h or 16 h while continuing with the same desiccating stress protocol. The fluorescein uptake test was performed following the indicated time period to assess the time length of CLU treatment effect. *p<0.0001 (n = 4)
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pone.0138958.g004: Topical CLU directly seals the ocular surface barrier disrupted by desiccating stress.The standard desiccating stress (DS) protocol was applied for 5-days to create ocular surface disruption. Non-stressed (NS) mice housed under normal ambient conditions served as a baseline control. Eyes with desiccating stress were then treated topically, a single time, with 1 uL of CLU formulated in PBS, 1 uL of BSA formulated in PBS for comparison, or 1 uL of PBS control. Barrier disruption was assayed by measuring corneal epithelial uptake of fluorescein (FU = Fluorescence Units at 521 nm). Values are expressed as the mean ± SD. (A) Eyes were treated a single time with recombinant human CLU (rhCLU) at 1, 3, 6 or 10 ug/mL, BSA at 10 ug/mL, or PBS. Fifteen minutes later, the fluorescein uptake test was performed, before there was time for barrier repair to occur. *P<0.0001 (n = 4). (B) Images of central cornea from the experiment shown in (A), obtained using laser scanning confocal microscopy at 10X magnification. One representative image out of two independent experiments is shown. Scale bar = 100 um. (C) Eyes were treated a single time with recombinant human CLU (rhCLU) at 10 ug/mL (right eyes) or PBS (left eyes). Then the mice were kept further for 2 h or 16 h while continuing with the same desiccating stress protocol. The fluorescein uptake test was performed following the indicated time period to assess the time length of CLU treatment effect. *p<0.0001 (n = 4)

Mentions: The amelioration results outlined above (Fig 3) suggested that one of the mechanisms of CLU action might be simply to seal areas of barrier damage so that dye can no longer penetrate. To test this idea, we applied the 5-day desiccating stress protocol, and then treated with CLU, but this time assayed for dye uptake within 15 minutes of treatment, giving the ocular surface no time to recover from the more severe stress (Fig 4A). An all-or-none response was observed once again, but the transition point was higher than when CLU was applied 4 times/day. Thus CLU at 6 ug/mL, applied one time, was completely effective in preventing dye uptake, while 3 ug/mL was completely ineffective. Laser scanning confocal microscopy was used to visualize punctate staining and its amelioration (Fig 4B). Eyes of mice subjected to desiccating stress and treated with BSA control showed many punctate spots of the size and shape of cells, similar to UT eyes, while desiccating stress eyes treated with CLU at 10 ug/mL showed far fewer spots, similar to the non-stressed control. In a second set of experiments we sought to determine how long the sealing effect would last. In a time course experiment, the sealing effect was maintained for 2 hours, but was lost by 16 hours (Fig 4C).


Clusterin Seals the Ocular Surface Barrier in Mouse Dry Eye.

Bauskar A, Mack WJ, Mauris J, Argüeso P, Heur M, Nagel BA, Kolar GR, Gleave ME, Nakamura T, Kinoshita S, Moradian-Oldak J, Panjwani N, Pflugfelder SC, Wilson MR, Fini ME, Jeong S - PLoS ONE (2015)

Topical CLU directly seals the ocular surface barrier disrupted by desiccating stress.The standard desiccating stress (DS) protocol was applied for 5-days to create ocular surface disruption. Non-stressed (NS) mice housed under normal ambient conditions served as a baseline control. Eyes with desiccating stress were then treated topically, a single time, with 1 uL of CLU formulated in PBS, 1 uL of BSA formulated in PBS for comparison, or 1 uL of PBS control. Barrier disruption was assayed by measuring corneal epithelial uptake of fluorescein (FU = Fluorescence Units at 521 nm). Values are expressed as the mean ± SD. (A) Eyes were treated a single time with recombinant human CLU (rhCLU) at 1, 3, 6 or 10 ug/mL, BSA at 10 ug/mL, or PBS. Fifteen minutes later, the fluorescein uptake test was performed, before there was time for barrier repair to occur. *P<0.0001 (n = 4). (B) Images of central cornea from the experiment shown in (A), obtained using laser scanning confocal microscopy at 10X magnification. One representative image out of two independent experiments is shown. Scale bar = 100 um. (C) Eyes were treated a single time with recombinant human CLU (rhCLU) at 10 ug/mL (right eyes) or PBS (left eyes). Then the mice were kept further for 2 h or 16 h while continuing with the same desiccating stress protocol. The fluorescein uptake test was performed following the indicated time period to assess the time length of CLU treatment effect. *p<0.0001 (n = 4)
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4581869&req=5

pone.0138958.g004: Topical CLU directly seals the ocular surface barrier disrupted by desiccating stress.The standard desiccating stress (DS) protocol was applied for 5-days to create ocular surface disruption. Non-stressed (NS) mice housed under normal ambient conditions served as a baseline control. Eyes with desiccating stress were then treated topically, a single time, with 1 uL of CLU formulated in PBS, 1 uL of BSA formulated in PBS for comparison, or 1 uL of PBS control. Barrier disruption was assayed by measuring corneal epithelial uptake of fluorescein (FU = Fluorescence Units at 521 nm). Values are expressed as the mean ± SD. (A) Eyes were treated a single time with recombinant human CLU (rhCLU) at 1, 3, 6 or 10 ug/mL, BSA at 10 ug/mL, or PBS. Fifteen minutes later, the fluorescein uptake test was performed, before there was time for barrier repair to occur. *P<0.0001 (n = 4). (B) Images of central cornea from the experiment shown in (A), obtained using laser scanning confocal microscopy at 10X magnification. One representative image out of two independent experiments is shown. Scale bar = 100 um. (C) Eyes were treated a single time with recombinant human CLU (rhCLU) at 10 ug/mL (right eyes) or PBS (left eyes). Then the mice were kept further for 2 h or 16 h while continuing with the same desiccating stress protocol. The fluorescein uptake test was performed following the indicated time period to assess the time length of CLU treatment effect. *p<0.0001 (n = 4)
Mentions: The amelioration results outlined above (Fig 3) suggested that one of the mechanisms of CLU action might be simply to seal areas of barrier damage so that dye can no longer penetrate. To test this idea, we applied the 5-day desiccating stress protocol, and then treated with CLU, but this time assayed for dye uptake within 15 minutes of treatment, giving the ocular surface no time to recover from the more severe stress (Fig 4A). An all-or-none response was observed once again, but the transition point was higher than when CLU was applied 4 times/day. Thus CLU at 6 ug/mL, applied one time, was completely effective in preventing dye uptake, while 3 ug/mL was completely ineffective. Laser scanning confocal microscopy was used to visualize punctate staining and its amelioration (Fig 4B). Eyes of mice subjected to desiccating stress and treated with BSA control showed many punctate spots of the size and shape of cells, similar to UT eyes, while desiccating stress eyes treated with CLU at 10 ug/mL showed far fewer spots, similar to the non-stressed control. In a second set of experiments we sought to determine how long the sealing effect would last. In a time course experiment, the sealing effect was maintained for 2 hours, but was lost by 16 hours (Fig 4C).

Bottom Line: When the CLU level drops below the critical all-or-none threshold, the barrier becomes vulnerable to desiccating stress.CLU binds selectively to the ocular surface subjected to desiccating stress in vivo, and in vitro to the galectin LGALS3, a key barrier component.Positioned in this way, CLU not only physically seals the ocular surface barrier, but it also protects the barrier cells and prevents further damage to barrier structure.

View Article: PubMed Central - PubMed

Affiliation: USC Institute for Genetic Medicine and Graduate Program in Medical Biology, Keck School of Medicine of USC, University of Southern California, Los Angeles, California, United States of America.

ABSTRACT
Dry eye is a common disorder caused by inadequate hydration of the ocular surface that results in disruption of barrier function. The homeostatic protein clusterin (CLU) is prominent at fluid-tissue interfaces throughout the body. CLU levels are reduced at the ocular surface in human inflammatory disorders that manifest as severe dry eye, as well as in a preclinical mouse model for desiccating stress that mimics dry eye. Using this mouse model, we show here that CLU prevents and ameliorates ocular surface barrier disruption by a remarkable sealing mechanism dependent on attainment of a critical all-or-none concentration. When the CLU level drops below the critical all-or-none threshold, the barrier becomes vulnerable to desiccating stress. CLU binds selectively to the ocular surface subjected to desiccating stress in vivo, and in vitro to the galectin LGALS3, a key barrier component. Positioned in this way, CLU not only physically seals the ocular surface barrier, but it also protects the barrier cells and prevents further damage to barrier structure. These findings define a fundamentally new mechanism for ocular surface protection and suggest CLU as a biotherapeutic for dry eye.

No MeSH data available.


Related in: MedlinePlus