Research
Original Investigation
Proteomic Landscape of the Human Choroid–Retinal Pigment Epithelial Complex Jessica M. Skeie, PhD; Vinit B. Mahajan, MD, PhD
IMPORTANCE Differences in geographical protein expression in the human choroid–retinal
pigment epithelial (RPE) complex may explain molecular predisposition of regions to ophthalmic diseases such as age-related macular degeneration.
Journal Club Slides and Supplemental content at jamaophthalmology.com
OBJECTIVE To characterize the proteome of the human choroid-RPE complex and to identify differentially expressed proteins in specific anatomic regions. DESIGN, SETTING, AND PARTICIPANTS Experimental study of choroid-RPE tissue from 3 nondiseased eyes. The choroid-RPE complex underwent biopsy from beneath the foveal, macular, and peripheral retina. Protein fractions were isolated and subjected to multidimensional liquid chromatography and tandem mass spectrometry. A bioinformatic pipeline matched peptide spectra to the human proteome, assigned gene ontology classification, and identified protein signaling pathways unique to each of the choroid-RPE regions. MAIN OUTCOMES AND MEASURES Mean number of mass spectra, statistically significant differentially expressed proteins, gene ontology classification, and pathway representation. RESULTS We identified a mean of 4403 unique proteins in each of the foveal, macular, and peripheral choroid-RPE tissues. Six hundred seventy-one differentially expressed proteins included previously known risk factors for retinal diseases related to oxidative stress, inflammation, and the complement cascade. Gene ontology analysis showed that unique categories in the foveal and macular regions included immune process proteins as well as protein complexes and plasma membrane proteins. The peripheral region contained unique antioxidant activity proteins. Many proteins had the highest expression in the foveal or macular regions, including inflammation-related proteins HLA-A, HLA-B, and HLA-C antigens; intercellular adhesion molecule 1 (ICAM-1); S100; transcription factor ERG; antioxidant superoxide dismutase 1 (SOD1); chloride intracellular channel 6 ion (CLIC6); activators of the complement cascade C1q, C6, and C8; and complement factor H. Proteins with higher expression in the periphery included bestrophin 1 (BEST1), transcription factor RNA binding motif protein 39 (RBM39), inflammatory mediator macrophage migration inhibitory factor, antioxidant SOD3, ion channel voltage-dependent anion-selective channel protein 3 (VDAC3), and complement inhibitor CD55. The complement activation was among the highest represented pathways (P < 7.5e−13). CONCLUSIONS AND RELEVANCE This proteomic data set identifies novel molecular signatures in anatomically sensitive regions of the choroid-RPE complex. The findings give mechanistic insight into choroid-RPE function, reveal important choroid-RPE processes, and prioritize new pathways for therapeutic targeting. Author Affiliations: Omics Laboratory, Department of Ophthalmology and Visual Sciences, University of Iowa Carver College of Medicine, Iowa City.
JAMA Ophthalmol. 2014;132(11):1271-1281. doi:10.1001/jamaophthalmol.2014.2065 Published online July 24, 2014.
Corresponding Author: Vinit B. Mahajan MD, PhD, Omics Laboratory, Department of Ophthalmology and Visual Sciences, University of Iowa Carver College of Medicine, 200 Hawkins Dr, Iowa City, IA 52242 ([email protected]).
1271
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Research Original Investigation
Proteomic Landscape of the Choroid-RPE Complex
T
he choroid is a high-flow vascular network with fenestrated capillaries that surround and nourish the neurosensory retina. Disruption of the choroid–retinal pigment epithelial (RPE) complex is a frequent cause of blinding retinal diseases. These diseases include age-related macular degeneration (AMD), central serous retinopathy, infectious and noninfectious chorioretinitis, and retinal degeneration.1 Many of these conditions localize to specific regions of the fundus. Neovascular AMD is frequently localized to the central foveal macula. Dry AMD, on the other hand, often spares the fovea. Infectious and noninfectious choroiditis occur throughout the peripheral fundus (Figure 1). Why choroidRPE diseases show regional susceptibility is not known. Anatomic variation of the choroid-RPE complex is one commonly proposed explanation for disease localization. 2-5 Higher metabolic demands imposed by the overlying retina compared with the periphery is another. We considered an alternative hypothesis that geographic differences in protein expression patterns could make some regions prone to disease. Several molecules are associated with choroid-RPE diseases. Complement and inflammatory molecules are strongly associated with AMD.6 Oxidative stress may also predispose the macula to AMD, because it can activate the innate immune response via the complement system.7-10 Glucocorticoids and ion channels are implicated in central serous retinopathy.11,12 Innate and adaptive immune responses are involved in chorioretinitis.13 Also, a number of molecules in the
outer retina rely on a functional interaction with the RPE and choroid, including those involved in energy metabolism, ion/ water transport, absorption of light, and waste removal.14 A comprehensive molecular map of the choroid-RPE complex could provide critical insight into disease mechanisms, but the normal distribution of these molecules across the choroidRPE complex has not been explored on a large scale. We used multidimensional liquid chromatography and tandem mass spectrometry in an unbiased systems biology approach to identify large numbers of proteins in regions of the choroid-RPE complex.15
Methods Human Choroid-RPE Tissue Collection This study was approved by the institutional review board of the University of Iowa and adheres to the tenets of the Declaration of Helsinki. Human donor tissue was obtained from the Iowa Lions Eye Bank, Iowa City. Tissue was obtained within 5 hours after death from 1 man and 2 women, all in their eighth and ninth decades of life. None of the eyes showed signs of retinal disease. Eyes were flowered into 4 quadrants as previously described.16 The choroid-RPE complex was collected using a 4-mm (fovea and periphery) or an 8-mm (macula around foveal punch) knife biopsy punch and stored in our biorepository until processed for mass spectrometry (Figure 1 and eFigure 1A in the Supplement).17
Figure 1. Fundus Images of Choroid–Retinal Pigment Epithelial (RPE) Complex Disease Display Region-Specific Diseases B
A
Fovea Macula Periphery
Macula
Periphery
Fovea
C
Fovea
D
A, Geographical map of the 3 different tissue samples. B, Detailed image of the human choroid-RPE tissue punch biopsy specimens collected for this study. C, Fundus image of neovascular age-related macular degeneration (arrowhead indicates subretinal hemorrhage from choroidal neovascularization; small circle,
1272
Macula
E
Periphery
fovea; large circle, macula area). D, Fundus image of macular geographic atrophy sparing the fovea (large circle indicates the macula area). E, Fundus image of pigment degeneration in the periphery.
JAMA Ophthalmology November 2014 Volume 132, Number 11
Copyright 2014 American Medical Association. All rights reserved.
Downloaded From: http://archopht.jamanetwork.com/ by a Universite Laval User on 05/11/2015
jamaophthalmology.com
Proteomic Landscape of the Choroid-RPE Complex
Mass Spectrometry Mass spectrometry was performed as previously described using a nanoflow system (Agilent 1100 series; Agilent Technologies) and a dual-pressure linear ion trap device (LTQ Velos, Thermo Fisher).18-20 The Mascot generic format (MGF) files were searched with X!Hunter21 against the latest library available in 2010 on the global proteome machine 22 and X!!Tandem23,24 using the native and k score25 algorithms and the open mass spectrometry search algorithm (OMSSA) (eFigure 1B-D in the Supplement).26
Bioinformatics Proteins were considered identified if they had an expectation value of less than 0.01 (ie,
Original Investigation
Proteomic Landscape of the Human Choroid–Retinal Pigment Epithelial Complex Jessica M. Skeie, PhD; Vinit B. Mahajan, MD, PhD
IMPORTANCE Differences in geographical protein expression in the human choroid–retinal
pigment epithelial (RPE) complex may explain molecular predisposition of regions to ophthalmic diseases such as age-related macular degeneration.
Journal Club Slides and Supplemental content at jamaophthalmology.com
OBJECTIVE To characterize the proteome of the human choroid-RPE complex and to identify differentially expressed proteins in specific anatomic regions. DESIGN, SETTING, AND PARTICIPANTS Experimental study of choroid-RPE tissue from 3 nondiseased eyes. The choroid-RPE complex underwent biopsy from beneath the foveal, macular, and peripheral retina. Protein fractions were isolated and subjected to multidimensional liquid chromatography and tandem mass spectrometry. A bioinformatic pipeline matched peptide spectra to the human proteome, assigned gene ontology classification, and identified protein signaling pathways unique to each of the choroid-RPE regions. MAIN OUTCOMES AND MEASURES Mean number of mass spectra, statistically significant differentially expressed proteins, gene ontology classification, and pathway representation. RESULTS We identified a mean of 4403 unique proteins in each of the foveal, macular, and peripheral choroid-RPE tissues. Six hundred seventy-one differentially expressed proteins included previously known risk factors for retinal diseases related to oxidative stress, inflammation, and the complement cascade. Gene ontology analysis showed that unique categories in the foveal and macular regions included immune process proteins as well as protein complexes and plasma membrane proteins. The peripheral region contained unique antioxidant activity proteins. Many proteins had the highest expression in the foveal or macular regions, including inflammation-related proteins HLA-A, HLA-B, and HLA-C antigens; intercellular adhesion molecule 1 (ICAM-1); S100; transcription factor ERG; antioxidant superoxide dismutase 1 (SOD1); chloride intracellular channel 6 ion (CLIC6); activators of the complement cascade C1q, C6, and C8; and complement factor H. Proteins with higher expression in the periphery included bestrophin 1 (BEST1), transcription factor RNA binding motif protein 39 (RBM39), inflammatory mediator macrophage migration inhibitory factor, antioxidant SOD3, ion channel voltage-dependent anion-selective channel protein 3 (VDAC3), and complement inhibitor CD55. The complement activation was among the highest represented pathways (P < 7.5e−13). CONCLUSIONS AND RELEVANCE This proteomic data set identifies novel molecular signatures in anatomically sensitive regions of the choroid-RPE complex. The findings give mechanistic insight into choroid-RPE function, reveal important choroid-RPE processes, and prioritize new pathways for therapeutic targeting. Author Affiliations: Omics Laboratory, Department of Ophthalmology and Visual Sciences, University of Iowa Carver College of Medicine, Iowa City.
JAMA Ophthalmol. 2014;132(11):1271-1281. doi:10.1001/jamaophthalmol.2014.2065 Published online July 24, 2014.
Corresponding Author: Vinit B. Mahajan MD, PhD, Omics Laboratory, Department of Ophthalmology and Visual Sciences, University of Iowa Carver College of Medicine, 200 Hawkins Dr, Iowa City, IA 52242 ([email protected]).
1271
Copyright 2014 American Medical Association. All rights reserved.
Downloaded From: http://archopht.jamanetwork.com/ by a Universite Laval User on 05/11/2015
Research Original Investigation
Proteomic Landscape of the Choroid-RPE Complex
T
he choroid is a high-flow vascular network with fenestrated capillaries that surround and nourish the neurosensory retina. Disruption of the choroid–retinal pigment epithelial (RPE) complex is a frequent cause of blinding retinal diseases. These diseases include age-related macular degeneration (AMD), central serous retinopathy, infectious and noninfectious chorioretinitis, and retinal degeneration.1 Many of these conditions localize to specific regions of the fundus. Neovascular AMD is frequently localized to the central foveal macula. Dry AMD, on the other hand, often spares the fovea. Infectious and noninfectious choroiditis occur throughout the peripheral fundus (Figure 1). Why choroidRPE diseases show regional susceptibility is not known. Anatomic variation of the choroid-RPE complex is one commonly proposed explanation for disease localization. 2-5 Higher metabolic demands imposed by the overlying retina compared with the periphery is another. We considered an alternative hypothesis that geographic differences in protein expression patterns could make some regions prone to disease. Several molecules are associated with choroid-RPE diseases. Complement and inflammatory molecules are strongly associated with AMD.6 Oxidative stress may also predispose the macula to AMD, because it can activate the innate immune response via the complement system.7-10 Glucocorticoids and ion channels are implicated in central serous retinopathy.11,12 Innate and adaptive immune responses are involved in chorioretinitis.13 Also, a number of molecules in the
outer retina rely on a functional interaction with the RPE and choroid, including those involved in energy metabolism, ion/ water transport, absorption of light, and waste removal.14 A comprehensive molecular map of the choroid-RPE complex could provide critical insight into disease mechanisms, but the normal distribution of these molecules across the choroidRPE complex has not been explored on a large scale. We used multidimensional liquid chromatography and tandem mass spectrometry in an unbiased systems biology approach to identify large numbers of proteins in regions of the choroid-RPE complex.15
Methods Human Choroid-RPE Tissue Collection This study was approved by the institutional review board of the University of Iowa and adheres to the tenets of the Declaration of Helsinki. Human donor tissue was obtained from the Iowa Lions Eye Bank, Iowa City. Tissue was obtained within 5 hours after death from 1 man and 2 women, all in their eighth and ninth decades of life. None of the eyes showed signs of retinal disease. Eyes were flowered into 4 quadrants as previously described.16 The choroid-RPE complex was collected using a 4-mm (fovea and periphery) or an 8-mm (macula around foveal punch) knife biopsy punch and stored in our biorepository until processed for mass spectrometry (Figure 1 and eFigure 1A in the Supplement).17
Figure 1. Fundus Images of Choroid–Retinal Pigment Epithelial (RPE) Complex Disease Display Region-Specific Diseases B
A
Fovea Macula Periphery
Macula
Periphery
Fovea
C
Fovea
D
A, Geographical map of the 3 different tissue samples. B, Detailed image of the human choroid-RPE tissue punch biopsy specimens collected for this study. C, Fundus image of neovascular age-related macular degeneration (arrowhead indicates subretinal hemorrhage from choroidal neovascularization; small circle,
1272
Macula
E
Periphery
fovea; large circle, macula area). D, Fundus image of macular geographic atrophy sparing the fovea (large circle indicates the macula area). E, Fundus image of pigment degeneration in the periphery.
JAMA Ophthalmology November 2014 Volume 132, Number 11
Copyright 2014 American Medical Association. All rights reserved.
Downloaded From: http://archopht.jamanetwork.com/ by a Universite Laval User on 05/11/2015
jamaophthalmology.com
Proteomic Landscape of the Choroid-RPE Complex
Mass Spectrometry Mass spectrometry was performed as previously described using a nanoflow system (Agilent 1100 series; Agilent Technologies) and a dual-pressure linear ion trap device (LTQ Velos, Thermo Fisher).18-20 The Mascot generic format (MGF) files were searched with X!Hunter21 against the latest library available in 2010 on the global proteome machine 22 and X!!Tandem23,24 using the native and k score25 algorithms and the open mass spectrometry search algorithm (OMSSA) (eFigure 1B-D in the Supplement).26
Bioinformatics Proteins were considered identified if they had an expectation value of less than 0.01 (ie,
