Effects of advanced glycation end-products (AGEs) on retinal pigment epithelial (RPE) cells lysosomal function: implications for age-related macular degeneration (AMD)

Luminita I. Paraoan, Umar Sharif, Paul Kay, Ian Grierson, Yit C. Yang, Simon P. Harding

Research output: Contribution to journalMeeting abstract

Abstract

Purpose: RPE lysosomal dysfunction is a major contributor to AMD pathogenesis. Controlled activity of a major class of RPE proteinases, the cathepsins, is crucial in maintaining correct lysosomal function. Advanced glycation end-products (AGEs) accumulate in the Bruch’s membrane (BM) with age, impacting critical RPE functions and in turn, contributing to the development of AMD. The aim of this study was to assess the effect of AGEs on lysosomal function by analysing the expression, processing and activity of the cysteine proteinases cathepsins B, L and S, and the aspartic proteinase cathepsin D.

Methods: ARPE-19 cells were cultured on AGE-containing BM mimics (matrigel) for 14 days and compared to untreated substrate. Expression levels and intracellular processing of cathepsins B, D, L and S, were assessed by qPCR and immunoblotting of cell lysates. Lysosomal activity was investigated using multiple activity assays specific to each of the analysed cathepsins. Statistical analysis was performed using the Student’s independent T-test.

Results: AGE exposure produced a 36% decrease in cathepsin L activity when compared to non-treated controls (p=0.02, n= 3) although no significant changes were observed in protein expression/processing under these conditions. Both the pro and active forms of cathepsin S decreased by 40% (p=0.04) and 74% (p=0.004), respectively (n=3). In contrast, the active form of the cathepsin D increased by 125% (p=0.005, n= 4). However, no changes were observed in the activity levels of both cathepsins S and D. In addition, cathepsin B expression, processing and activity also remained unaltered following AGE exposure.

Conclusions: AGEs accumulation in the extracellular matrix, a phenomenon associated with the natural aging process of the BM, attenuates the expression, intracellular processing and activity of specific lysosomal effectors. Altered enzymatic function may impair important lysosomal processes such as endocytosis, autophagy and phagocytosis of photoreceptor outer segments, each of which may influence the age-related dysfunction of the RPE and subsequently, AMD pathogenesis.

Original languageEnglish
Pages (from-to)4197
Number of pages1
JournalInvestigative Ophthalmology and Visual Science
Volume56
Issue number7
Publication statusPublished - 30 Jun 2015
EventARVO 2015 Annual Meeting : Powerful Connections: Vision Research and Online Networking - Colorado Convention Center (CCC), Denver, CO, United States
Duration: 2 May 20157 May 2015

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cathepsin S
Advanced Glycosylation End Products
Retinal Pigments
Macular Degeneration
Cathepsin D
Epithelial Cells
Bruch Membrane
Cathepsin B
Cathepsin L
Cathepsins
yeast proteinase B
Aspartic Acid Proteases
Cysteine Proteases
Autophagy
Endocytosis
Phagocytosis
Immunoblotting
Extracellular Matrix
Cultured Cells
Peptide Hydrolases

Bibliographical note

ARVO Annual Meeting Abstract: Annual Meeting of the Association-for-Research-in-Vision-and-Ophthalmology (ARVO)

Cite this

@article{23f7702ccf644243b8c66f1e6ba18fc8,
title = "Effects of advanced glycation end-products (AGEs) on retinal pigment epithelial (RPE) cells lysosomal function: implications for age-related macular degeneration (AMD)",
abstract = "Purpose: RPE lysosomal dysfunction is a major contributor to AMD pathogenesis. Controlled activity of a major class of RPE proteinases, the cathepsins, is crucial in maintaining correct lysosomal function. Advanced glycation end-products (AGEs) accumulate in the Bruch’s membrane (BM) with age, impacting critical RPE functions and in turn, contributing to the development of AMD. The aim of this study was to assess the effect of AGEs on lysosomal function by analysing the expression, processing and activity of the cysteine proteinases cathepsins B, L and S, and the aspartic proteinase cathepsin D.Methods: ARPE-19 cells were cultured on AGE-containing BM mimics (matrigel) for 14 days and compared to untreated substrate. Expression levels and intracellular processing of cathepsins B, D, L and S, were assessed by qPCR and immunoblotting of cell lysates. Lysosomal activity was investigated using multiple activity assays specific to each of the analysed cathepsins. Statistical analysis was performed using the Student’s independent T-test.Results: AGE exposure produced a 36{\%} decrease in cathepsin L activity when compared to non-treated controls (p=0.02, n= 3) although no significant changes were observed in protein expression/processing under these conditions. Both the pro and active forms of cathepsin S decreased by 40{\%} (p=0.04) and 74{\%} (p=0.004), respectively (n=3). In contrast, the active form of the cathepsin D increased by 125{\%} (p=0.005, n= 4). However, no changes were observed in the activity levels of both cathepsins S and D. In addition, cathepsin B expression, processing and activity also remained unaltered following AGE exposure.Conclusions: AGEs accumulation in the extracellular matrix, a phenomenon associated with the natural aging process of the BM, attenuates the expression, intracellular processing and activity of specific lysosomal effectors. Altered enzymatic function may impair important lysosomal processes such as endocytosis, autophagy and phagocytosis of photoreceptor outer segments, each of which may influence the age-related dysfunction of the RPE and subsequently, AMD pathogenesis.",
author = "Paraoan, {Luminita I.} and Umar Sharif and Paul Kay and Ian Grierson and Yang, {Yit C.} and Harding, {Simon P.}",
note = "ARVO Annual Meeting Abstract: Annual Meeting of the Association-for-Research-in-Vision-and-Ophthalmology (ARVO)",
year = "2015",
month = "6",
day = "30",
language = "English",
volume = "56",
pages = "4197",
journal = "Investigative Ophthalmology and Visual Science",
issn = "1552-5783",
publisher = "Association for Research in Vision and Ophthalmology Inc.",
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Effects of advanced glycation end-products (AGEs) on retinal pigment epithelial (RPE) cells lysosomal function : implications for age-related macular degeneration (AMD). / Paraoan, Luminita I.; Sharif, Umar; Kay, Paul; Grierson, Ian; Yang, Yit C.; Harding, Simon P.

In: Investigative Ophthalmology and Visual Science, Vol. 56, No. 7, 30.06.2015, p. 4197.

Research output: Contribution to journalMeeting abstract

TY - JOUR

T1 - Effects of advanced glycation end-products (AGEs) on retinal pigment epithelial (RPE) cells lysosomal function

T2 - implications for age-related macular degeneration (AMD)

AU - Paraoan, Luminita I.

AU - Sharif, Umar

AU - Kay, Paul

AU - Grierson, Ian

AU - Yang, Yit C.

AU - Harding, Simon P.

N1 - ARVO Annual Meeting Abstract: Annual Meeting of the Association-for-Research-in-Vision-and-Ophthalmology (ARVO)

PY - 2015/6/30

Y1 - 2015/6/30

N2 - Purpose: RPE lysosomal dysfunction is a major contributor to AMD pathogenesis. Controlled activity of a major class of RPE proteinases, the cathepsins, is crucial in maintaining correct lysosomal function. Advanced glycation end-products (AGEs) accumulate in the Bruch’s membrane (BM) with age, impacting critical RPE functions and in turn, contributing to the development of AMD. The aim of this study was to assess the effect of AGEs on lysosomal function by analysing the expression, processing and activity of the cysteine proteinases cathepsins B, L and S, and the aspartic proteinase cathepsin D.Methods: ARPE-19 cells were cultured on AGE-containing BM mimics (matrigel) for 14 days and compared to untreated substrate. Expression levels and intracellular processing of cathepsins B, D, L and S, were assessed by qPCR and immunoblotting of cell lysates. Lysosomal activity was investigated using multiple activity assays specific to each of the analysed cathepsins. Statistical analysis was performed using the Student’s independent T-test.Results: AGE exposure produced a 36% decrease in cathepsin L activity when compared to non-treated controls (p=0.02, n= 3) although no significant changes were observed in protein expression/processing under these conditions. Both the pro and active forms of cathepsin S decreased by 40% (p=0.04) and 74% (p=0.004), respectively (n=3). In contrast, the active form of the cathepsin D increased by 125% (p=0.005, n= 4). However, no changes were observed in the activity levels of both cathepsins S and D. In addition, cathepsin B expression, processing and activity also remained unaltered following AGE exposure.Conclusions: AGEs accumulation in the extracellular matrix, a phenomenon associated with the natural aging process of the BM, attenuates the expression, intracellular processing and activity of specific lysosomal effectors. Altered enzymatic function may impair important lysosomal processes such as endocytosis, autophagy and phagocytosis of photoreceptor outer segments, each of which may influence the age-related dysfunction of the RPE and subsequently, AMD pathogenesis.

AB - Purpose: RPE lysosomal dysfunction is a major contributor to AMD pathogenesis. Controlled activity of a major class of RPE proteinases, the cathepsins, is crucial in maintaining correct lysosomal function. Advanced glycation end-products (AGEs) accumulate in the Bruch’s membrane (BM) with age, impacting critical RPE functions and in turn, contributing to the development of AMD. The aim of this study was to assess the effect of AGEs on lysosomal function by analysing the expression, processing and activity of the cysteine proteinases cathepsins B, L and S, and the aspartic proteinase cathepsin D.Methods: ARPE-19 cells were cultured on AGE-containing BM mimics (matrigel) for 14 days and compared to untreated substrate. Expression levels and intracellular processing of cathepsins B, D, L and S, were assessed by qPCR and immunoblotting of cell lysates. Lysosomal activity was investigated using multiple activity assays specific to each of the analysed cathepsins. Statistical analysis was performed using the Student’s independent T-test.Results: AGE exposure produced a 36% decrease in cathepsin L activity when compared to non-treated controls (p=0.02, n= 3) although no significant changes were observed in protein expression/processing under these conditions. Both the pro and active forms of cathepsin S decreased by 40% (p=0.04) and 74% (p=0.004), respectively (n=3). In contrast, the active form of the cathepsin D increased by 125% (p=0.005, n= 4). However, no changes were observed in the activity levels of both cathepsins S and D. In addition, cathepsin B expression, processing and activity also remained unaltered following AGE exposure.Conclusions: AGEs accumulation in the extracellular matrix, a phenomenon associated with the natural aging process of the BM, attenuates the expression, intracellular processing and activity of specific lysosomal effectors. Altered enzymatic function may impair important lysosomal processes such as endocytosis, autophagy and phagocytosis of photoreceptor outer segments, each of which may influence the age-related dysfunction of the RPE and subsequently, AMD pathogenesis.

M3 - Meeting abstract

VL - 56

SP - 4197

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

SN - 1552-5783

IS - 7

ER -