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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 protects the ocular surface barrier via an all-or-none mechanism.The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically 4 times/day with 1 uL of CLU formulated in PBS, or with PBS control. Non-stressed (NS) mice housed under normal ambient conditions served as a baseline control. After the indicated time period, barrier integrity was assayed by measuring corneal epithelial uptake of fluorescein (FU = Fluorescence Units at 521 nm). Values are expressed as the mean ± SD. (A) Dose response experiment. The desiccating stress (DS) protocol was applied for 5 days while also treating with (Left) recombinant human CLU (rhCLU) at the indicated 10-fold dilutions (n = 6), (Middle) recombinant human CLU (rhCLU) at 0.1, 0.3, 0.6, or 1 ug/mL (n = 6), or (Right) recombinant mouse CLU (rmCLU) at 0.3, 0.6, and 1 ug/mL (n = 4). *P<0.0001. (B) Experiment comparing CLU with BSA. The desiccating stress (DS) protocol was applied for 5 days while also treating with recombinant human CLU (rhCLU) and BSA, individually or in combination, as indicated. *P<0.0001 (n = 4). (C) Stress reduction experiment. The standard desiccating stress (DS) protocol was applied for 5 days while eyes were also treated with recombinant human CLU (rhCLU) at 0.01, 0.1, and 1 ug/mL. Using a subset (n = 4) of each treatment group the effect of each rhCLU dose on integrity of the ocular surface barrier was confirmed by the fluorescein uptake test at day 5. Then the rest of the mice in each treatment group were subjected for two more days to a more moderate desiccating stress by continuing with the air draft and heat, but omitting scopolamine and CLU treatments. The fluorescein uptake test was then performed on these remaining mice. *P = 0.004 (n = 4); **P = 0.05 (n = 4)
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pone.0138958.g002: Topical CLU protects the ocular surface barrier via an all-or-none mechanism.The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically 4 times/day with 1 uL of CLU formulated in PBS, or with PBS control. Non-stressed (NS) mice housed under normal ambient conditions served as a baseline control. After the indicated time period, barrier integrity was assayed by measuring corneal epithelial uptake of fluorescein (FU = Fluorescence Units at 521 nm). Values are expressed as the mean ± SD. (A) Dose response experiment. The desiccating stress (DS) protocol was applied for 5 days while also treating with (Left) recombinant human CLU (rhCLU) at the indicated 10-fold dilutions (n = 6), (Middle) recombinant human CLU (rhCLU) at 0.1, 0.3, 0.6, or 1 ug/mL (n = 6), or (Right) recombinant mouse CLU (rmCLU) at 0.3, 0.6, and 1 ug/mL (n = 4). *P<0.0001. (B) Experiment comparing CLU with BSA. The desiccating stress (DS) protocol was applied for 5 days while also treating with recombinant human CLU (rhCLU) and BSA, individually or in combination, as indicated. *P<0.0001 (n = 4). (C) Stress reduction experiment. The standard desiccating stress (DS) protocol was applied for 5 days while eyes were also treated with recombinant human CLU (rhCLU) at 0.01, 0.1, and 1 ug/mL. Using a subset (n = 4) of each treatment group the effect of each rhCLU dose on integrity of the ocular surface barrier was confirmed by the fluorescein uptake test at day 5. Then the rest of the mice in each treatment group were subjected for two more days to a more moderate desiccating stress by continuing with the air draft and heat, but omitting scopolamine and CLU treatments. The fluorescein uptake test was then performed on these remaining mice. *P = 0.004 (n = 4); **P = 0.05 (n = 4)

Mentions: To determine a dose-response for barrier protection by CLU, we next applied the 5-day desiccating stress protocol while simultaneously treating the ocular surface with serial 10-fold dilutions of rhCLU. Similar to results of the experiment shown above (Fig 1), treatment with 1 ug/mL or 10 ug/mL almost completely protected against fluorescein uptake. In contrast, lower concentrations had essentially no effect, with values similar to UT and PBS-treated groups (Fig 2A Left). To determine any gradation in activity between 0.1 and 1 ug/mL CLU, we tested CLU concentrations at tight intervals in between these doses (Fig 2A Middle). We observed a transition in effectiveness between 0.6 ug/mL and 1 ug/mL, essentially an all-or-none response. We also tested rmCLU; the dose transition was at exactly the same place, between 0.6 and 1 ug/mL (Fig 2A Right). Next, we tested whether BSA, as an in vitro protein stabilizer and as a non-CLU protein also found in serum, could enhance the protective activity of CLU at the low concentration. BSA did not show any significant protective or enhancing effect, alone or with CLU at 0.6 ug/mL, compared with 1 ug/mL of CLU alone (Fig 2B). Use of Alexa-Fluor-dextran in the fluorescein uptake assay, which is more discriminating because of its much larger molecular size [14], gave identical results (data not shown).


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 protects the ocular surface barrier via an all-or-none mechanism.The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically 4 times/day with 1 uL of CLU formulated in PBS, or with PBS control. Non-stressed (NS) mice housed under normal ambient conditions served as a baseline control. After the indicated time period, barrier integrity was assayed by measuring corneal epithelial uptake of fluorescein (FU = Fluorescence Units at 521 nm). Values are expressed as the mean ± SD. (A) Dose response experiment. The desiccating stress (DS) protocol was applied for 5 days while also treating with (Left) recombinant human CLU (rhCLU) at the indicated 10-fold dilutions (n = 6), (Middle) recombinant human CLU (rhCLU) at 0.1, 0.3, 0.6, or 1 ug/mL (n = 6), or (Right) recombinant mouse CLU (rmCLU) at 0.3, 0.6, and 1 ug/mL (n = 4). *P<0.0001. (B) Experiment comparing CLU with BSA. The desiccating stress (DS) protocol was applied for 5 days while also treating with recombinant human CLU (rhCLU) and BSA, individually or in combination, as indicated. *P<0.0001 (n = 4). (C) Stress reduction experiment. The standard desiccating stress (DS) protocol was applied for 5 days while eyes were also treated with recombinant human CLU (rhCLU) at 0.01, 0.1, and 1 ug/mL. Using a subset (n = 4) of each treatment group the effect of each rhCLU dose on integrity of the ocular surface barrier was confirmed by the fluorescein uptake test at day 5. Then the rest of the mice in each treatment group were subjected for two more days to a more moderate desiccating stress by continuing with the air draft and heat, but omitting scopolamine and CLU treatments. The fluorescein uptake test was then performed on these remaining mice. *P = 0.004 (n = 4); **P = 0.05 (n = 4)
© Copyright Policy
Related In: Results  -  Collection

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pone.0138958.g002: Topical CLU protects the ocular surface barrier via an all-or-none mechanism.The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically 4 times/day with 1 uL of CLU formulated in PBS, or with PBS control. Non-stressed (NS) mice housed under normal ambient conditions served as a baseline control. After the indicated time period, barrier integrity was assayed by measuring corneal epithelial uptake of fluorescein (FU = Fluorescence Units at 521 nm). Values are expressed as the mean ± SD. (A) Dose response experiment. The desiccating stress (DS) protocol was applied for 5 days while also treating with (Left) recombinant human CLU (rhCLU) at the indicated 10-fold dilutions (n = 6), (Middle) recombinant human CLU (rhCLU) at 0.1, 0.3, 0.6, or 1 ug/mL (n = 6), or (Right) recombinant mouse CLU (rmCLU) at 0.3, 0.6, and 1 ug/mL (n = 4). *P<0.0001. (B) Experiment comparing CLU with BSA. The desiccating stress (DS) protocol was applied for 5 days while also treating with recombinant human CLU (rhCLU) and BSA, individually or in combination, as indicated. *P<0.0001 (n = 4). (C) Stress reduction experiment. The standard desiccating stress (DS) protocol was applied for 5 days while eyes were also treated with recombinant human CLU (rhCLU) at 0.01, 0.1, and 1 ug/mL. Using a subset (n = 4) of each treatment group the effect of each rhCLU dose on integrity of the ocular surface barrier was confirmed by the fluorescein uptake test at day 5. Then the rest of the mice in each treatment group were subjected for two more days to a more moderate desiccating stress by continuing with the air draft and heat, but omitting scopolamine and CLU treatments. The fluorescein uptake test was then performed on these remaining mice. *P = 0.004 (n = 4); **P = 0.05 (n = 4)
Mentions: To determine a dose-response for barrier protection by CLU, we next applied the 5-day desiccating stress protocol while simultaneously treating the ocular surface with serial 10-fold dilutions of rhCLU. Similar to results of the experiment shown above (Fig 1), treatment with 1 ug/mL or 10 ug/mL almost completely protected against fluorescein uptake. In contrast, lower concentrations had essentially no effect, with values similar to UT and PBS-treated groups (Fig 2A Left). To determine any gradation in activity between 0.1 and 1 ug/mL CLU, we tested CLU concentrations at tight intervals in between these doses (Fig 2A Middle). We observed a transition in effectiveness between 0.6 ug/mL and 1 ug/mL, essentially an all-or-none response. We also tested rmCLU; the dose transition was at exactly the same place, between 0.6 and 1 ug/mL (Fig 2A Right). Next, we tested whether BSA, as an in vitro protein stabilizer and as a non-CLU protein also found in serum, could enhance the protective activity of CLU at the low concentration. BSA did not show any significant protective or enhancing effect, alone or with CLU at 0.6 ug/mL, compared with 1 ug/mL of CLU alone (Fig 2B). Use of Alexa-Fluor-dextran in the fluorescein uptake assay, which is more discriminating because of its much larger molecular size [14], gave identical results (data not shown).

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