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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 proteolytic damage due to desiccating stress.(A) The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically, 4 times/day, with 1 uL of recombinant human CLU (rhCLU) formulated in PBS, or with 1 uL of PBS control. Non-stressed (NS) mice housed under normal ambient conditions were included as a control for PBS treatment. At the end of the experiment, eyes were removed and embedded for frozen sectioning at 10-um thickness. TUNEL staining was performed and nuclei were counterstained with DAPI. Images were taken at 20X magnification. Arrows indicate apoptotic cells in the apical ocular surface epithelium of DS+PBS eyes. (B) The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically, 4 times/day, with 1 uL of recombinant human CLU (rhCLU) formulated in PBS, or with 1 uL of PBS control. Non-stressed (NS) mice housed under normal ambient conditions were included as a control for PBS treatment. Desiccating stress was applied to 7 mice per treatment group for 5 days (OCLN) or 9 days (LGALS3) while treated with PBS or CLU at 1 ug/mL. Then total proteins were extracted from the ocular surface epithelia using TRIzol, pooled among the same treatment groups, and subjected to Western blotting with anti-LGALS3 and anti-OCLN antibodies. The protein band image was obtained by Fuji Doc digital camera. “F” indicates full length LGALS3 protein, and “C” is the cleaved product of LGALS3. A digital image analyzer built into the camera was used to quantify the density of individual protein bands. The relative cleavage of LGALS3 was calculated by ratio of the C over the total (F+C) LGALS3 protein. The relative amount of OCLN was normalized to the loading control (ACTB) in each gel lane. (C) Stratified HCLE cells were treated with TNFA (5 ng/mL), alone or with recombinant human CLU (rhCLU) (4 ug/mL) or BSA (40 ug/mL) for 24 h. the conditioned media were subject to gelatin zymography and the developed MMP9 image were analyzed by Image J software. *P<0.05 (n = 3, student’s t-test)
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pone.0138958.g006: Topical CLU protects the ocular surface barrier against proteolytic damage due to desiccating stress.(A) The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically, 4 times/day, with 1 uL of recombinant human CLU (rhCLU) formulated in PBS, or with 1 uL of PBS control. Non-stressed (NS) mice housed under normal ambient conditions were included as a control for PBS treatment. At the end of the experiment, eyes were removed and embedded for frozen sectioning at 10-um thickness. TUNEL staining was performed and nuclei were counterstained with DAPI. Images were taken at 20X magnification. Arrows indicate apoptotic cells in the apical ocular surface epithelium of DS+PBS eyes. (B) The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically, 4 times/day, with 1 uL of recombinant human CLU (rhCLU) formulated in PBS, or with 1 uL of PBS control. Non-stressed (NS) mice housed under normal ambient conditions were included as a control for PBS treatment. Desiccating stress was applied to 7 mice per treatment group for 5 days (OCLN) or 9 days (LGALS3) while treated with PBS or CLU at 1 ug/mL. Then total proteins were extracted from the ocular surface epithelia using TRIzol, pooled among the same treatment groups, and subjected to Western blotting with anti-LGALS3 and anti-OCLN antibodies. The protein band image was obtained by Fuji Doc digital camera. “F” indicates full length LGALS3 protein, and “C” is the cleaved product of LGALS3. A digital image analyzer built into the camera was used to quantify the density of individual protein bands. The relative cleavage of LGALS3 was calculated by ratio of the C over the total (F+C) LGALS3 protein. The relative amount of OCLN was normalized to the loading control (ACTB) in each gel lane. (C) Stratified HCLE cells were treated with TNFA (5 ng/mL), alone or with recombinant human CLU (rhCLU) (4 ug/mL) or BSA (40 ug/mL) for 24 h. the conditioned media were subject to gelatin zymography and the developed MMP9 image were analyzed by Image J software. *P<0.05 (n = 3, student’s t-test)

Mentions: Having demonstrated the capacity of CLU to protect the ocular surface barrier against functional disruption due to desiccating stress, we next tested its capacity to protect the cells and proteins of the barrier against physical damage. First we investigated the cytoprotective activity of topical CLU. Representative results are shown in Fig 6A. Only a few cells at the ocular surface of non-stressed (NS) eyes were positively stained in the TUNEL assay, a measure of DNA damage characteristic of apoptotic cells. In the PBS-treated DS eye, staining of epithelial cells and stroma cells was strikingly increased, consistent with previous observations [11–13, 50]. However, when the ocular surface was treated topically with CLU at the same time as it was subjected to desiccating stress, the level of TUNEL staining remained the same as in non-stressed eyes.


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 proteolytic damage due to desiccating stress.(A) The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically, 4 times/day, with 1 uL of recombinant human CLU (rhCLU) formulated in PBS, or with 1 uL of PBS control. Non-stressed (NS) mice housed under normal ambient conditions were included as a control for PBS treatment. At the end of the experiment, eyes were removed and embedded for frozen sectioning at 10-um thickness. TUNEL staining was performed and nuclei were counterstained with DAPI. Images were taken at 20X magnification. Arrows indicate apoptotic cells in the apical ocular surface epithelium of DS+PBS eyes. (B) The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically, 4 times/day, with 1 uL of recombinant human CLU (rhCLU) formulated in PBS, or with 1 uL of PBS control. Non-stressed (NS) mice housed under normal ambient conditions were included as a control for PBS treatment. Desiccating stress was applied to 7 mice per treatment group for 5 days (OCLN) or 9 days (LGALS3) while treated with PBS or CLU at 1 ug/mL. Then total proteins were extracted from the ocular surface epithelia using TRIzol, pooled among the same treatment groups, and subjected to Western blotting with anti-LGALS3 and anti-OCLN antibodies. The protein band image was obtained by Fuji Doc digital camera. “F” indicates full length LGALS3 protein, and “C” is the cleaved product of LGALS3. A digital image analyzer built into the camera was used to quantify the density of individual protein bands. The relative cleavage of LGALS3 was calculated by ratio of the C over the total (F+C) LGALS3 protein. The relative amount of OCLN was normalized to the loading control (ACTB) in each gel lane. (C) Stratified HCLE cells were treated with TNFA (5 ng/mL), alone or with recombinant human CLU (rhCLU) (4 ug/mL) or BSA (40 ug/mL) for 24 h. the conditioned media were subject to gelatin zymography and the developed MMP9 image were analyzed by Image J software. *P<0.05 (n = 3, student’s t-test)
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4581869&req=5

pone.0138958.g006: Topical CLU protects the ocular surface barrier against proteolytic damage due to desiccating stress.(A) The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically, 4 times/day, with 1 uL of recombinant human CLU (rhCLU) formulated in PBS, or with 1 uL of PBS control. Non-stressed (NS) mice housed under normal ambient conditions were included as a control for PBS treatment. At the end of the experiment, eyes were removed and embedded for frozen sectioning at 10-um thickness. TUNEL staining was performed and nuclei were counterstained with DAPI. Images were taken at 20X magnification. Arrows indicate apoptotic cells in the apical ocular surface epithelium of DS+PBS eyes. (B) The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically, 4 times/day, with 1 uL of recombinant human CLU (rhCLU) formulated in PBS, or with 1 uL of PBS control. Non-stressed (NS) mice housed under normal ambient conditions were included as a control for PBS treatment. Desiccating stress was applied to 7 mice per treatment group for 5 days (OCLN) or 9 days (LGALS3) while treated with PBS or CLU at 1 ug/mL. Then total proteins were extracted from the ocular surface epithelia using TRIzol, pooled among the same treatment groups, and subjected to Western blotting with anti-LGALS3 and anti-OCLN antibodies. The protein band image was obtained by Fuji Doc digital camera. “F” indicates full length LGALS3 protein, and “C” is the cleaved product of LGALS3. A digital image analyzer built into the camera was used to quantify the density of individual protein bands. The relative cleavage of LGALS3 was calculated by ratio of the C over the total (F+C) LGALS3 protein. The relative amount of OCLN was normalized to the loading control (ACTB) in each gel lane. (C) Stratified HCLE cells were treated with TNFA (5 ng/mL), alone or with recombinant human CLU (rhCLU) (4 ug/mL) or BSA (40 ug/mL) for 24 h. the conditioned media were subject to gelatin zymography and the developed MMP9 image were analyzed by Image J software. *P<0.05 (n = 3, student’s t-test)
Mentions: Having demonstrated the capacity of CLU to protect the ocular surface barrier against functional disruption due to desiccating stress, we next tested its capacity to protect the cells and proteins of the barrier against physical damage. First we investigated the cytoprotective activity of topical CLU. Representative results are shown in Fig 6A. Only a few cells at the ocular surface of non-stressed (NS) eyes were positively stained in the TUNEL assay, a measure of DNA damage characteristic of apoptotic cells. In the PBS-treated DS eye, staining of epithelial cells and stroma cells was strikingly increased, consistent with previous observations [11–13, 50]. However, when the ocular surface was treated topically with CLU at the same time as it was subjected to desiccating stress, the level of TUNEL staining remained the same as in non-stressed eyes.

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