Repetitive nonlethal oxidant injury to retinal pigment epithelium decreased extracellular matrix turnover in vitro and induced Sub-RPE deposits in vivo

Maria E. Marin-Castaño, Gary E. Striker, Oscar Alcazar, Paola Catanuto, Diego Gabriel Espinosa Heidmann, Scott W. Cousins

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

PURPOSE. To determine the impact of repetitive nonlethal oxidant injury with hydroquinone (HQ) on regulation of cell membrane blebbing and molecules, which are essential in extracellular matrix turnover (ECM) maintenance, especially matrix metalloproteinase (MMP)-2, tissue inhibitor of MMP (TIMP)-2, and type IV collagen in cultured RPE. In addition, to determine whether chronic oral HQ causes induction of sub-RPE deposit formation in a mouse model. METHODS. An ARPE-19 cell line stably expressing membrane-targeted green fluorescent protein (GFP) was challenged by exposure to HQ (100 μM). Repetitive acute (6 hours every 3 days for 4 weeks) or transient (6 hours followed by a recovery phase, every 5 days for 6 weeks) exposure to HQ were evaluated. An MTS assay, cell counts, and bromodeoxyuridine (BrdU) incorporation were used to detect cell viability and proliferation. Supernatants and cell homogenates were collected to assess MMP-2 and TIMP-2 activity by zymography and reverse zymography, proteins by Western blot, and type IV collagen accumulation by ELISA and immunostaining. Expression of MMP-2 and type IV collagen was examined by real-time RT-PCR on total RNA. Sixteen-month-old C57BL/6 female mice were fed a regular fat diet, with or without HQ (0.8%) in the drinking water, for 4 months. The eyes were removed for transmission electron microscopy of the retina and choroid after treatment. Semiquantitative grading of deposit severity was performed. RESULTS. In vitro, high doses of HQ (400-250 μM) killed a significant fraction of RPE cells (∼60% of control). Low doses (50-100 μM) were nonlethal but induced significant blebbing. Both nonlethal repetitive acute and transient exposure to HQ were associated with diminished MMP-2 activity and increased collagen type IV accumulation. In vivo, mice exposed to oral HQ demonstrated moderately thick basal laminar deposits and a variable degree of deposits within Bruch's membrane (BrM). These homogeneous sub-RPE deposits accumulated in the eyes, consistent with early laminar deposits. CONCLUSIONS. In cultured RPE, nonlethal injury with HQ upregulated nonlethal blebbing and decreased ECM turnover. Similarly, in vivo exposure to oral HQ induced nonlethal bleb injury and sub-RPE deposits. These data support the hypothesis that HQ may regulate blebbing and molecules that influence ECM turnover. This study suggests that HQ may be another type of oxidant that causes injury to the RPE and may explain the association between environmental oxidants and early AMD.

Original languageEnglish (US)
Pages (from-to)4098-4112
Number of pages15
JournalInvestigative Ophthalmology and Visual Science
Volume47
Issue number9
DOIs
StatePublished - Sep 1 2006
Externally publishedYes

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Retinal Pigment Epithelium
Oxidants
Extracellular Matrix
Wounds and Injuries
Matrix Metalloproteinase 2
Blister
Collagen Type IV
hydroquinone
In Vitro Techniques
Bruch Membrane
Tissue Inhibitor of Metalloproteinase-2
Matrix Metalloproteinase Inhibitors
Choroid
Bromodeoxyuridine
Green Fluorescent Proteins
Matrix Metalloproteinases
Transmission Electron Microscopy
Drinking Water
Retina
Real-Time Polymerase Chain Reaction

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Repetitive nonlethal oxidant injury to retinal pigment epithelium decreased extracellular matrix turnover in vitro and induced Sub-RPE deposits in vivo. / Marin-Castaño, Maria E.; Striker, Gary E.; Alcazar, Oscar; Catanuto, Paola; Espinosa Heidmann, Diego Gabriel; Cousins, Scott W.

In: Investigative Ophthalmology and Visual Science, Vol. 47, No. 9, 01.09.2006, p. 4098-4112.

Research output: Contribution to journalArticle

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abstract = "PURPOSE. To determine the impact of repetitive nonlethal oxidant injury with hydroquinone (HQ) on regulation of cell membrane blebbing and molecules, which are essential in extracellular matrix turnover (ECM) maintenance, especially matrix metalloproteinase (MMP)-2, tissue inhibitor of MMP (TIMP)-2, and type IV collagen in cultured RPE. In addition, to determine whether chronic oral HQ causes induction of sub-RPE deposit formation in a mouse model. METHODS. An ARPE-19 cell line stably expressing membrane-targeted green fluorescent protein (GFP) was challenged by exposure to HQ (100 μM). Repetitive acute (6 hours every 3 days for 4 weeks) or transient (6 hours followed by a recovery phase, every 5 days for 6 weeks) exposure to HQ were evaluated. An MTS assay, cell counts, and bromodeoxyuridine (BrdU) incorporation were used to detect cell viability and proliferation. Supernatants and cell homogenates were collected to assess MMP-2 and TIMP-2 activity by zymography and reverse zymography, proteins by Western blot, and type IV collagen accumulation by ELISA and immunostaining. Expression of MMP-2 and type IV collagen was examined by real-time RT-PCR on total RNA. Sixteen-month-old C57BL/6 female mice were fed a regular fat diet, with or without HQ (0.8{\%}) in the drinking water, for 4 months. The eyes were removed for transmission electron microscopy of the retina and choroid after treatment. Semiquantitative grading of deposit severity was performed. RESULTS. In vitro, high doses of HQ (400-250 μM) killed a significant fraction of RPE cells (∼60{\%} of control). Low doses (50-100 μM) were nonlethal but induced significant blebbing. Both nonlethal repetitive acute and transient exposure to HQ were associated with diminished MMP-2 activity and increased collagen type IV accumulation. In vivo, mice exposed to oral HQ demonstrated moderately thick basal laminar deposits and a variable degree of deposits within Bruch's membrane (BrM). These homogeneous sub-RPE deposits accumulated in the eyes, consistent with early laminar deposits. CONCLUSIONS. In cultured RPE, nonlethal injury with HQ upregulated nonlethal blebbing and decreased ECM turnover. Similarly, in vivo exposure to oral HQ induced nonlethal bleb injury and sub-RPE deposits. These data support the hypothesis that HQ may regulate blebbing and molecules that influence ECM turnover. This study suggests that HQ may be another type of oxidant that causes injury to the RPE and may explain the association between environmental oxidants and early AMD.",
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T1 - Repetitive nonlethal oxidant injury to retinal pigment epithelium decreased extracellular matrix turnover in vitro and induced Sub-RPE deposits in vivo

AU - Marin-Castaño, Maria E.

AU - Striker, Gary E.

AU - Alcazar, Oscar

AU - Catanuto, Paola

AU - Espinosa Heidmann, Diego Gabriel

AU - Cousins, Scott W.

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N2 - PURPOSE. To determine the impact of repetitive nonlethal oxidant injury with hydroquinone (HQ) on regulation of cell membrane blebbing and molecules, which are essential in extracellular matrix turnover (ECM) maintenance, especially matrix metalloproteinase (MMP)-2, tissue inhibitor of MMP (TIMP)-2, and type IV collagen in cultured RPE. In addition, to determine whether chronic oral HQ causes induction of sub-RPE deposit formation in a mouse model. METHODS. An ARPE-19 cell line stably expressing membrane-targeted green fluorescent protein (GFP) was challenged by exposure to HQ (100 μM). Repetitive acute (6 hours every 3 days for 4 weeks) or transient (6 hours followed by a recovery phase, every 5 days for 6 weeks) exposure to HQ were evaluated. An MTS assay, cell counts, and bromodeoxyuridine (BrdU) incorporation were used to detect cell viability and proliferation. Supernatants and cell homogenates were collected to assess MMP-2 and TIMP-2 activity by zymography and reverse zymography, proteins by Western blot, and type IV collagen accumulation by ELISA and immunostaining. Expression of MMP-2 and type IV collagen was examined by real-time RT-PCR on total RNA. Sixteen-month-old C57BL/6 female mice were fed a regular fat diet, with or without HQ (0.8%) in the drinking water, for 4 months. The eyes were removed for transmission electron microscopy of the retina and choroid after treatment. Semiquantitative grading of deposit severity was performed. RESULTS. In vitro, high doses of HQ (400-250 μM) killed a significant fraction of RPE cells (∼60% of control). Low doses (50-100 μM) were nonlethal but induced significant blebbing. Both nonlethal repetitive acute and transient exposure to HQ were associated with diminished MMP-2 activity and increased collagen type IV accumulation. In vivo, mice exposed to oral HQ demonstrated moderately thick basal laminar deposits and a variable degree of deposits within Bruch's membrane (BrM). These homogeneous sub-RPE deposits accumulated in the eyes, consistent with early laminar deposits. CONCLUSIONS. In cultured RPE, nonlethal injury with HQ upregulated nonlethal blebbing and decreased ECM turnover. Similarly, in vivo exposure to oral HQ induced nonlethal bleb injury and sub-RPE deposits. These data support the hypothesis that HQ may regulate blebbing and molecules that influence ECM turnover. This study suggests that HQ may be another type of oxidant that causes injury to the RPE and may explain the association between environmental oxidants and early AMD.

AB - PURPOSE. To determine the impact of repetitive nonlethal oxidant injury with hydroquinone (HQ) on regulation of cell membrane blebbing and molecules, which are essential in extracellular matrix turnover (ECM) maintenance, especially matrix metalloproteinase (MMP)-2, tissue inhibitor of MMP (TIMP)-2, and type IV collagen in cultured RPE. In addition, to determine whether chronic oral HQ causes induction of sub-RPE deposit formation in a mouse model. METHODS. An ARPE-19 cell line stably expressing membrane-targeted green fluorescent protein (GFP) was challenged by exposure to HQ (100 μM). Repetitive acute (6 hours every 3 days for 4 weeks) or transient (6 hours followed by a recovery phase, every 5 days for 6 weeks) exposure to HQ were evaluated. An MTS assay, cell counts, and bromodeoxyuridine (BrdU) incorporation were used to detect cell viability and proliferation. Supernatants and cell homogenates were collected to assess MMP-2 and TIMP-2 activity by zymography and reverse zymography, proteins by Western blot, and type IV collagen accumulation by ELISA and immunostaining. Expression of MMP-2 and type IV collagen was examined by real-time RT-PCR on total RNA. Sixteen-month-old C57BL/6 female mice were fed a regular fat diet, with or without HQ (0.8%) in the drinking water, for 4 months. The eyes were removed for transmission electron microscopy of the retina and choroid after treatment. Semiquantitative grading of deposit severity was performed. RESULTS. In vitro, high doses of HQ (400-250 μM) killed a significant fraction of RPE cells (∼60% of control). Low doses (50-100 μM) were nonlethal but induced significant blebbing. Both nonlethal repetitive acute and transient exposure to HQ were associated with diminished MMP-2 activity and increased collagen type IV accumulation. In vivo, mice exposed to oral HQ demonstrated moderately thick basal laminar deposits and a variable degree of deposits within Bruch's membrane (BrM). These homogeneous sub-RPE deposits accumulated in the eyes, consistent with early laminar deposits. CONCLUSIONS. In cultured RPE, nonlethal injury with HQ upregulated nonlethal blebbing and decreased ECM turnover. Similarly, in vivo exposure to oral HQ induced nonlethal bleb injury and sub-RPE deposits. These data support the hypothesis that HQ may regulate blebbing and molecules that influence ECM turnover. This study suggests that HQ may be another type of oxidant that causes injury to the RPE and may explain the association between environmental oxidants and early AMD.

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