Mutant fibulin-3 causes proteoglycan accumulation and impaired diffusion across bruch’s membrane

Astrid Zayas-Santiago, Samuel D. Cross, James B. Stanton, Alan D Marmorstein, Lihua Y Marmorstein

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

PURPOSE. The mutation R345W in EFEMP1 (fibulin-3) causes macular degeneration. This study sought to determine whether proteoglycan content and diffusion across Bruch’s membrane are altered in Efemp1ki/ki mice carrying this mutation or in Efemp1-/- mice. METHODS. Proteoglycans in mouse Bruch’s membranes were stained with Cupromeronic Blue (CB). Heparan sulfated proteoglycan (HSPG) and chondroitin/dermatan sulfate proteoglycan (C/DSPG) distributions were visualized following treatments with chondroitinase ABC (C-ABC) or nitrous acid. Total sulfated glycosaminoglycans (sGAGs) in Bruch’s membrane/ choroid (BrM/Ch) were measured with dimethylmethylene blue (DMMB). Matrix metalloprotease (MMP)-2, MMP-9, and tissue inhibitor of metalloproteinase (TIMP)-3 were examined by immunofluorescence and quantified using Image J. Molecules with different Stokes radius (Rs) were allowed simultaneously to diffuse through mouse BrM/Ch mounted in a modified Ussing chamber. Samples were quantified using gel exclusion chromatography. RESULTS. HSPGs and C/DSPGs were markedly increased in Efemp1ki/ki Bruch’s membrane, and MMP-2 and MMP-9 were decreased, but TIMP-3 was increased. Diffusion across Efemp1ki/ki Bruch’s membrane was impaired. In contrast, the proteoglycan amount in Efemp1-/- Bruch’s membrane was not significantly different, but the size of proteoglycans was much larger. MMP-2, MMP-3, and TIMP-3 levels were similar to that of Efemp1+/+ mice, but they were localized diffusely in retinal pigment epithelium (RPE) cells instead of Bruch’s membrane. Diffusion across Efemp1-/- Bruch’s membrane was enhanced. CONCLUSIONS. Mutant fibulin-3 causes proteoglycan accumulation, reduction of MMP-2 and MMP-9, but increase of TIMP-3, and impairs diffusion across Bruch’s membrane. Fibulin-3 ablation results in altered sizes of proteoglycans, altered distributions of MMP-2, MMP-9, and TIMP-3, and enhances diffusion across Bruch’s membrane.

Original languageEnglish (US)
Pages (from-to)3046-3054
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume58
Issue number7
DOIs
StatePublished - Jun 1 2017

Fingerprint

Bruch Membrane
Proteoglycans
Metalloproteases
Tissue Inhibitor of Metalloproteinase-3
Choroid
A73025
Gel Chromatography
fibulin
Nitrous Acid
Chondroitin ABC Lyase
Chondroitin Sulfate Proteoglycans
Heparan Sulfate Proteoglycans
Mutation
Retinal Pigment Epithelium
Macular Degeneration
Fluorescent Antibody Technique

Keywords

  • Age-related macular degeneration
  • Diffusion
  • EFEMP1 (fibulin-3)
  • Malattia Leventinese
  • Proteoglycans

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Mutant fibulin-3 causes proteoglycan accumulation and impaired diffusion across bruch’s membrane. / Zayas-Santiago, Astrid; Cross, Samuel D.; Stanton, James B.; Marmorstein, Alan D; Marmorstein, Lihua Y.

In: Investigative Ophthalmology and Visual Science, Vol. 58, No. 7, 01.06.2017, p. 3046-3054.

Research output: Contribution to journalArticle

Zayas-Santiago, Astrid ; Cross, Samuel D. ; Stanton, James B. ; Marmorstein, Alan D ; Marmorstein, Lihua Y. / Mutant fibulin-3 causes proteoglycan accumulation and impaired diffusion across bruch’s membrane. In: Investigative Ophthalmology and Visual Science. 2017 ; Vol. 58, No. 7. pp. 3046-3054.
@article{48ae42f08e2f43139b25b3d8007e0d9e,
title = "Mutant fibulin-3 causes proteoglycan accumulation and impaired diffusion across bruch’s membrane",
abstract = "PURPOSE. The mutation R345W in EFEMP1 (fibulin-3) causes macular degeneration. This study sought to determine whether proteoglycan content and diffusion across Bruch’s membrane are altered in Efemp1ki/ki mice carrying this mutation or in Efemp1-/- mice. METHODS. Proteoglycans in mouse Bruch’s membranes were stained with Cupromeronic Blue (CB). Heparan sulfated proteoglycan (HSPG) and chondroitin/dermatan sulfate proteoglycan (C/DSPG) distributions were visualized following treatments with chondroitinase ABC (C-ABC) or nitrous acid. Total sulfated glycosaminoglycans (sGAGs) in Bruch’s membrane/ choroid (BrM/Ch) were measured with dimethylmethylene blue (DMMB). Matrix metalloprotease (MMP)-2, MMP-9, and tissue inhibitor of metalloproteinase (TIMP)-3 were examined by immunofluorescence and quantified using Image J. Molecules with different Stokes radius (Rs) were allowed simultaneously to diffuse through mouse BrM/Ch mounted in a modified Ussing chamber. Samples were quantified using gel exclusion chromatography. RESULTS. HSPGs and C/DSPGs were markedly increased in Efemp1ki/ki Bruch’s membrane, and MMP-2 and MMP-9 were decreased, but TIMP-3 was increased. Diffusion across Efemp1ki/ki Bruch’s membrane was impaired. In contrast, the proteoglycan amount in Efemp1-/- Bruch’s membrane was not significantly different, but the size of proteoglycans was much larger. MMP-2, MMP-3, and TIMP-3 levels were similar to that of Efemp1+/+ mice, but they were localized diffusely in retinal pigment epithelium (RPE) cells instead of Bruch’s membrane. Diffusion across Efemp1-/- Bruch’s membrane was enhanced. CONCLUSIONS. Mutant fibulin-3 causes proteoglycan accumulation, reduction of MMP-2 and MMP-9, but increase of TIMP-3, and impairs diffusion across Bruch’s membrane. Fibulin-3 ablation results in altered sizes of proteoglycans, altered distributions of MMP-2, MMP-9, and TIMP-3, and enhances diffusion across Bruch’s membrane.",
keywords = "Age-related macular degeneration, Diffusion, EFEMP1 (fibulin-3), Malattia Leventinese, Proteoglycans",
author = "Astrid Zayas-Santiago and Cross, {Samuel D.} and Stanton, {James B.} and Marmorstein, {Alan D} and Marmorstein, {Lihua Y}",
year = "2017",
month = "6",
day = "1",
doi = "10.1167/iovs.17-21720",
language = "English (US)",
volume = "58",
pages = "3046--3054",
journal = "Investigative Ophthalmology and Visual Science",
issn = "0146-0404",
publisher = "Association for Research in Vision and Ophthalmology Inc.",
number = "7",

}

TY - JOUR

T1 - Mutant fibulin-3 causes proteoglycan accumulation and impaired diffusion across bruch’s membrane

AU - Zayas-Santiago, Astrid

AU - Cross, Samuel D.

AU - Stanton, James B.

AU - Marmorstein, Alan D

AU - Marmorstein, Lihua Y

PY - 2017/6/1

Y1 - 2017/6/1

N2 - PURPOSE. The mutation R345W in EFEMP1 (fibulin-3) causes macular degeneration. This study sought to determine whether proteoglycan content and diffusion across Bruch’s membrane are altered in Efemp1ki/ki mice carrying this mutation or in Efemp1-/- mice. METHODS. Proteoglycans in mouse Bruch’s membranes were stained with Cupromeronic Blue (CB). Heparan sulfated proteoglycan (HSPG) and chondroitin/dermatan sulfate proteoglycan (C/DSPG) distributions were visualized following treatments with chondroitinase ABC (C-ABC) or nitrous acid. Total sulfated glycosaminoglycans (sGAGs) in Bruch’s membrane/ choroid (BrM/Ch) were measured with dimethylmethylene blue (DMMB). Matrix metalloprotease (MMP)-2, MMP-9, and tissue inhibitor of metalloproteinase (TIMP)-3 were examined by immunofluorescence and quantified using Image J. Molecules with different Stokes radius (Rs) were allowed simultaneously to diffuse through mouse BrM/Ch mounted in a modified Ussing chamber. Samples were quantified using gel exclusion chromatography. RESULTS. HSPGs and C/DSPGs were markedly increased in Efemp1ki/ki Bruch’s membrane, and MMP-2 and MMP-9 were decreased, but TIMP-3 was increased. Diffusion across Efemp1ki/ki Bruch’s membrane was impaired. In contrast, the proteoglycan amount in Efemp1-/- Bruch’s membrane was not significantly different, but the size of proteoglycans was much larger. MMP-2, MMP-3, and TIMP-3 levels were similar to that of Efemp1+/+ mice, but they were localized diffusely in retinal pigment epithelium (RPE) cells instead of Bruch’s membrane. Diffusion across Efemp1-/- Bruch’s membrane was enhanced. CONCLUSIONS. Mutant fibulin-3 causes proteoglycan accumulation, reduction of MMP-2 and MMP-9, but increase of TIMP-3, and impairs diffusion across Bruch’s membrane. Fibulin-3 ablation results in altered sizes of proteoglycans, altered distributions of MMP-2, MMP-9, and TIMP-3, and enhances diffusion across Bruch’s membrane.

AB - PURPOSE. The mutation R345W in EFEMP1 (fibulin-3) causes macular degeneration. This study sought to determine whether proteoglycan content and diffusion across Bruch’s membrane are altered in Efemp1ki/ki mice carrying this mutation or in Efemp1-/- mice. METHODS. Proteoglycans in mouse Bruch’s membranes were stained with Cupromeronic Blue (CB). Heparan sulfated proteoglycan (HSPG) and chondroitin/dermatan sulfate proteoglycan (C/DSPG) distributions were visualized following treatments with chondroitinase ABC (C-ABC) or nitrous acid. Total sulfated glycosaminoglycans (sGAGs) in Bruch’s membrane/ choroid (BrM/Ch) were measured with dimethylmethylene blue (DMMB). Matrix metalloprotease (MMP)-2, MMP-9, and tissue inhibitor of metalloproteinase (TIMP)-3 were examined by immunofluorescence and quantified using Image J. Molecules with different Stokes radius (Rs) were allowed simultaneously to diffuse through mouse BrM/Ch mounted in a modified Ussing chamber. Samples were quantified using gel exclusion chromatography. RESULTS. HSPGs and C/DSPGs were markedly increased in Efemp1ki/ki Bruch’s membrane, and MMP-2 and MMP-9 were decreased, but TIMP-3 was increased. Diffusion across Efemp1ki/ki Bruch’s membrane was impaired. In contrast, the proteoglycan amount in Efemp1-/- Bruch’s membrane was not significantly different, but the size of proteoglycans was much larger. MMP-2, MMP-3, and TIMP-3 levels were similar to that of Efemp1+/+ mice, but they were localized diffusely in retinal pigment epithelium (RPE) cells instead of Bruch’s membrane. Diffusion across Efemp1-/- Bruch’s membrane was enhanced. CONCLUSIONS. Mutant fibulin-3 causes proteoglycan accumulation, reduction of MMP-2 and MMP-9, but increase of TIMP-3, and impairs diffusion across Bruch’s membrane. Fibulin-3 ablation results in altered sizes of proteoglycans, altered distributions of MMP-2, MMP-9, and TIMP-3, and enhances diffusion across Bruch’s membrane.

KW - Age-related macular degeneration

KW - Diffusion

KW - EFEMP1 (fibulin-3)

KW - Malattia Leventinese

KW - Proteoglycans

UR - http://www.scopus.com/inward/record.url?scp=85020909288&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85020909288&partnerID=8YFLogxK

U2 - 10.1167/iovs.17-21720

DO - 10.1167/iovs.17-21720

M3 - Article

VL - 58

SP - 3046

EP - 3054

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

SN - 0146-0404

IS - 7

ER -