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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 against functional disruption by desiccating stress.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) The desiccating stress (DS) protocol was applied for 5 days while also treating with rhCLU at 10 or 100 ug/mL. *P<0.0001 (n = 9). (B) The desiccating stress (DS) protocol was applied for 7 days while also treating with rhCLU at 1 or 10 ug/mL. *P<0.0001 (n = 4). (C) The desiccating stress (DS) protocol was applied for 5 days while also treating with human plasma CLU (pCLU) at 2 ug/mL *P<0.0001 (n = 4). (D) The desiccating stress (DS) protocol was applied for 5 days while also treating with recombinant mouse CLU (rmCLU) at 2 ug/mL. *P<0.0001 (n = 4)
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pone.0138958.g001: Topical CLU protects the ocular surface barrier against functional disruption by desiccating stress.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) The desiccating stress (DS) protocol was applied for 5 days while also treating with rhCLU at 10 or 100 ug/mL. *P<0.0001 (n = 9). (B) The desiccating stress (DS) protocol was applied for 7 days while also treating with rhCLU at 1 or 10 ug/mL. *P<0.0001 (n = 4). (C) The desiccating stress (DS) protocol was applied for 5 days while also treating with human plasma CLU (pCLU) at 2 ug/mL *P<0.0001 (n = 4). (D) The desiccating stress (DS) protocol was applied for 5 days while also treating with recombinant mouse CLU (rmCLU) at 2 ug/mL. *P<0.0001 (n = 4)

Mentions: To determine whether supplementation with topical CLU could protect against disruption of the ocular surface barrier subjected to desiccating stress, we applied the 5-day desiccating stress protocol to mice, and also treated topically with recombinant human CLU (rhCLU) formulated in PBS, applied 4 times/day at the same time as scopolamine was administered. After 5 days, barrier integrity was quantified by measuring uptake of fluorescein dye. Results were compared to controls treated with PBS vehicle alone. The stressed but untreated (UT) ocular surface served as the control for PBS treatment and non-stressed (NS) eyes served as the baseline control. Since CLU concentration in human serum was known to be in the range of 100±50 ug/mL [49], we used 10 or 100 ug/mL of rhCLU for our first experiments (Fig 1A). Dye uptake in stressed eyes treated with PBS alone was ~8-fold greater than NS counterparts. In contrast, dye uptake in eyes that were stressed, while also being treated with CLU at 10 or 100 ug/mL, was similar to that of NS counterparts, indicating complete protection against barrier disruption. We performed a second set of experiments using a 7-day desiccating stress protocol and rhCLU concentrations of 1 and 10 ug/mL. Again we observed nearly complete protection against barrier disruption as measured by dye uptake at both concentrations (Fig 1B). We performed a similar experiment using a 5-day desiccating stress protocol, but using human plasma CLU (pCLU) (Fig 1C) or recombinant mouse CLU (rmCLU) (Fig 1D) to rule out the possibility that the results might be unique to rhCLU. Treatment with 2 ug/mL of pCLU or rmCLU consistently protected against barrier disruption as measured by fluorescein uptake, to the same extent as rhCLU at 2 ug/mL, and was comparable to NS controls.


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 against functional disruption by desiccating stress.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) The desiccating stress (DS) protocol was applied for 5 days while also treating with rhCLU at 10 or 100 ug/mL. *P<0.0001 (n = 9). (B) The desiccating stress (DS) protocol was applied for 7 days while also treating with rhCLU at 1 or 10 ug/mL. *P<0.0001 (n = 4). (C) The desiccating stress (DS) protocol was applied for 5 days while also treating with human plasma CLU (pCLU) at 2 ug/mL *P<0.0001 (n = 4). (D) The desiccating stress (DS) protocol was applied for 5 days while also treating with recombinant mouse CLU (rmCLU) at 2 ug/mL. *P<0.0001 (n = 4)
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4581869&req=5

pone.0138958.g001: Topical CLU protects the ocular surface barrier against functional disruption by desiccating stress.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) The desiccating stress (DS) protocol was applied for 5 days while also treating with rhCLU at 10 or 100 ug/mL. *P<0.0001 (n = 9). (B) The desiccating stress (DS) protocol was applied for 7 days while also treating with rhCLU at 1 or 10 ug/mL. *P<0.0001 (n = 4). (C) The desiccating stress (DS) protocol was applied for 5 days while also treating with human plasma CLU (pCLU) at 2 ug/mL *P<0.0001 (n = 4). (D) The desiccating stress (DS) protocol was applied for 5 days while also treating with recombinant mouse CLU (rmCLU) at 2 ug/mL. *P<0.0001 (n = 4)
Mentions: To determine whether supplementation with topical CLU could protect against disruption of the ocular surface barrier subjected to desiccating stress, we applied the 5-day desiccating stress protocol to mice, and also treated topically with recombinant human CLU (rhCLU) formulated in PBS, applied 4 times/day at the same time as scopolamine was administered. After 5 days, barrier integrity was quantified by measuring uptake of fluorescein dye. Results were compared to controls treated with PBS vehicle alone. The stressed but untreated (UT) ocular surface served as the control for PBS treatment and non-stressed (NS) eyes served as the baseline control. Since CLU concentration in human serum was known to be in the range of 100±50 ug/mL [49], we used 10 or 100 ug/mL of rhCLU for our first experiments (Fig 1A). Dye uptake in stressed eyes treated with PBS alone was ~8-fold greater than NS counterparts. In contrast, dye uptake in eyes that were stressed, while also being treated with CLU at 10 or 100 ug/mL, was similar to that of NS counterparts, indicating complete protection against barrier disruption. We performed a second set of experiments using a 7-day desiccating stress protocol and rhCLU concentrations of 1 and 10 ug/mL. Again we observed nearly complete protection against barrier disruption as measured by dye uptake at both concentrations (Fig 1B). We performed a similar experiment using a 5-day desiccating stress protocol, but using human plasma CLU (pCLU) (Fig 1C) or recombinant mouse CLU (rmCLU) (Fig 1D) to rule out the possibility that the results might be unique to rhCLU. Treatment with 2 ug/mL of pCLU or rmCLU consistently protected against barrier disruption as measured by fluorescein uptake, to the same extent as rhCLU at 2 ug/mL, and was comparable to NS controls.

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