Selective permeability of mouse blood-aqueous barrier as determined by15N-heavy isotope tracing and mass spectrometry

Pan Liu, Benjamin R. Thomson, Natalia Khalatyan, Liang Feng, Xiaorong Liu, Jeffrey Nicholas Savas, Susan E Quaggin, Jing Jin*

*Corresponding author for this work

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

The blood-aqueous barrier plays a key role in regulating aqueous humor homeostasis by selectively restricting passage of proteins into the eye. The kinetics of aqueous flow are traditionally measured using artificial markers; however, these marker molecules do not address the barrier’s selective permeability to plasma proteins. Here we applied stable isotope labeling of all serum proteins with nitrogen-15 (15N) atoms. Following systemic injection of this “heavy” serum in mice, the 15N-to-endogenous nitrogen-14 (14N) ratio of each protein in aqueous was measured by mass spectrometry. By monitoring the kinetic changes in these ratios, we determined the permeability profiles of hundreds of serum proteins. Meanwhile, we subjected one of the eyes to neoangiogenic wound healing by inflicting injury to the corneal limbus and compared the 15N proteomes between the normal eyes and the recovering eyes at 2 weeks after injury. In the injured eye, we detected markedly enhanced permeability to inhibitory complement regulator proteins, such as Cfh, Cfhr, Cfb, Cfi, Cfd, and Vtn. Many of the proteins in this group are implicated in age-related macular degeneration associated with leakage of the blood-retinal barrier due to inflammation. To rule out the possibility that the observed leakage was due simply to physical damage of the blood vessels, we separately created a neovascularization model using an alkali burn of the avascular cornea. In this latter model, elevated levels of Cfh and Cfb were evident. These findings suggest that ocular neovascularization is associated with enhanced permeability to serum complement regulators.

Original languageEnglish (US)
Pages (from-to)9032-9037
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume115
Issue number36
DOIs
StatePublished - Sep 4 2018

Fingerprint

Blood-Aqueous Barrier
Isotopes
Permeability
Mass Spectrometry
Blood Proteins
Eye Proteins
Nitrogen
Limbus Corneae
Blood-Retinal Barrier
Isotope Labeling
Aqueous Humor
Wounds and Injuries
Macular Degeneration
Alkalies
Proteome
Serum
Wound Healing
Cornea
Blood Vessels
Complement System Proteins

Keywords

  • Blood-aqueous barrier
  • Blood-ocular barrier
  • Complement system
  • Labeling in mammals
  • Proteomics
  • Stable isotope

ASJC Scopus subject areas

  • General

Cite this

@article{f455a108c854441da28ba840aef75cd7,
title = "Selective permeability of mouse blood-aqueous barrier as determined by15N-heavy isotope tracing and mass spectrometry",
abstract = "The blood-aqueous barrier plays a key role in regulating aqueous humor homeostasis by selectively restricting passage of proteins into the eye. The kinetics of aqueous flow are traditionally measured using artificial markers; however, these marker molecules do not address the barrier’s selective permeability to plasma proteins. Here we applied stable isotope labeling of all serum proteins with nitrogen-15 (15N) atoms. Following systemic injection of this “heavy” serum in mice, the 15N-to-endogenous nitrogen-14 (14N) ratio of each protein in aqueous was measured by mass spectrometry. By monitoring the kinetic changes in these ratios, we determined the permeability profiles of hundreds of serum proteins. Meanwhile, we subjected one of the eyes to neoangiogenic wound healing by inflicting injury to the corneal limbus and compared the 15N proteomes between the normal eyes and the recovering eyes at 2 weeks after injury. In the injured eye, we detected markedly enhanced permeability to inhibitory complement regulator proteins, such as Cfh, Cfhr, Cfb, Cfi, Cfd, and Vtn. Many of the proteins in this group are implicated in age-related macular degeneration associated with leakage of the blood-retinal barrier due to inflammation. To rule out the possibility that the observed leakage was due simply to physical damage of the blood vessels, we separately created a neovascularization model using an alkali burn of the avascular cornea. In this latter model, elevated levels of Cfh and Cfb were evident. These findings suggest that ocular neovascularization is associated with enhanced permeability to serum complement regulators.",
keywords = "Blood-aqueous barrier, Blood-ocular barrier, Complement system, Labeling in mammals, Proteomics, Stable isotope",
author = "Pan Liu and Thomson, {Benjamin R.} and Natalia Khalatyan and Liang Feng and Xiaorong Liu and Savas, {Jeffrey Nicholas} and Quaggin, {Susan E} and Jing Jin",
year = "2018",
month = "9",
day = "4",
doi = "10.1073/pnas.1807982115",
language = "English (US)",
volume = "115",
pages = "9032--9037",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "36",

}

Selective permeability of mouse blood-aqueous barrier as determined by15N-heavy isotope tracing and mass spectrometry. / Liu, Pan; Thomson, Benjamin R.; Khalatyan, Natalia; Feng, Liang; Liu, Xiaorong; Savas, Jeffrey Nicholas; Quaggin, Susan E; Jin, Jing.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 115, No. 36, 04.09.2018, p. 9032-9037.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Selective permeability of mouse blood-aqueous barrier as determined by15N-heavy isotope tracing and mass spectrometry

AU - Liu, Pan

AU - Thomson, Benjamin R.

AU - Khalatyan, Natalia

AU - Feng, Liang

AU - Liu, Xiaorong

AU - Savas, Jeffrey Nicholas

AU - Quaggin, Susan E

AU - Jin, Jing

PY - 2018/9/4

Y1 - 2018/9/4

N2 - The blood-aqueous barrier plays a key role in regulating aqueous humor homeostasis by selectively restricting passage of proteins into the eye. The kinetics of aqueous flow are traditionally measured using artificial markers; however, these marker molecules do not address the barrier’s selective permeability to plasma proteins. Here we applied stable isotope labeling of all serum proteins with nitrogen-15 (15N) atoms. Following systemic injection of this “heavy” serum in mice, the 15N-to-endogenous nitrogen-14 (14N) ratio of each protein in aqueous was measured by mass spectrometry. By monitoring the kinetic changes in these ratios, we determined the permeability profiles of hundreds of serum proteins. Meanwhile, we subjected one of the eyes to neoangiogenic wound healing by inflicting injury to the corneal limbus and compared the 15N proteomes between the normal eyes and the recovering eyes at 2 weeks after injury. In the injured eye, we detected markedly enhanced permeability to inhibitory complement regulator proteins, such as Cfh, Cfhr, Cfb, Cfi, Cfd, and Vtn. Many of the proteins in this group are implicated in age-related macular degeneration associated with leakage of the blood-retinal barrier due to inflammation. To rule out the possibility that the observed leakage was due simply to physical damage of the blood vessels, we separately created a neovascularization model using an alkali burn of the avascular cornea. In this latter model, elevated levels of Cfh and Cfb were evident. These findings suggest that ocular neovascularization is associated with enhanced permeability to serum complement regulators.

AB - The blood-aqueous barrier plays a key role in regulating aqueous humor homeostasis by selectively restricting passage of proteins into the eye. The kinetics of aqueous flow are traditionally measured using artificial markers; however, these marker molecules do not address the barrier’s selective permeability to plasma proteins. Here we applied stable isotope labeling of all serum proteins with nitrogen-15 (15N) atoms. Following systemic injection of this “heavy” serum in mice, the 15N-to-endogenous nitrogen-14 (14N) ratio of each protein in aqueous was measured by mass spectrometry. By monitoring the kinetic changes in these ratios, we determined the permeability profiles of hundreds of serum proteins. Meanwhile, we subjected one of the eyes to neoangiogenic wound healing by inflicting injury to the corneal limbus and compared the 15N proteomes between the normal eyes and the recovering eyes at 2 weeks after injury. In the injured eye, we detected markedly enhanced permeability to inhibitory complement regulator proteins, such as Cfh, Cfhr, Cfb, Cfi, Cfd, and Vtn. Many of the proteins in this group are implicated in age-related macular degeneration associated with leakage of the blood-retinal barrier due to inflammation. To rule out the possibility that the observed leakage was due simply to physical damage of the blood vessels, we separately created a neovascularization model using an alkali burn of the avascular cornea. In this latter model, elevated levels of Cfh and Cfb were evident. These findings suggest that ocular neovascularization is associated with enhanced permeability to serum complement regulators.

KW - Blood-aqueous barrier

KW - Blood-ocular barrier

KW - Complement system

KW - Labeling in mammals

KW - Proteomics

KW - Stable isotope

UR - http://www.scopus.com/inward/record.url?scp=85052737562&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85052737562&partnerID=8YFLogxK

U2 - 10.1073/pnas.1807982115

DO - 10.1073/pnas.1807982115

M3 - Article

VL - 115

SP - 9032

EP - 9037

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 36

ER -