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Proteomics in the Vitreous of Diabetic Retinopathy Patients
Edward P. Feener
CONTENTS
INTRODUCTION
VITREOUS ANATOMY
A CANDIDATE APPROACH
PROTEOMIC APPROACHES
THE VITREOUS PROTEOME
SUMMARY AND CONCLUSIONS
ACKNOWLEDGMENTS
REFERENCES
Keywords Diabetic retinopathy • Mass spectrometry • Proteomics • Retina • Vitreous
INTRODUCTION
Vision loss cause by diabetic retinopathy is primarily associated with advanced stages of this disease, including proliferative diabetic retinopathy (PDR) and diabetic macular edema (DME). While abnormalities in microvascular functions and structure appear central to the progression of diabetic retinopathy [1], the specific factors that modulate the transition to the advanced sight-threatening stages of this disease are not fully understood. Moreover, since animal models do not reproduce many of the specific pathologies associated with PDR and DME, further characterization of ocular biochemical changes from patients with diabetic retinopathy is needed to identify factors that could be associated with the advance stages of this disease and vision loss. Analyses of vitreous fluid obtained during pars plana vitrectomy have provided opportunities to identify factors that may contribute to, or protect against, advanced stages of diabetic retinopathy. This chapter examines the methodologies for vitreous proteomics and the findings that are beginning to emerge from studies using this approach.
Characterization of vitreous from patients with diabetic retinopathy compared with vitreous from nondiabetic subjects has revealed a variety of differences in intraocular
From: Ophthalmology Research: Visual Dysfunction in Diabetes
Edited by: J. Tombran-Tink et al. (eds.), DOI 10.1007/978-1-60761-150-9_11 © Springer Science+Business Media, LLC 2012
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protein abundance, modification, and activities. Over the past several decades, a variety of biochemical and immunological techniques have been used to characterize specific candidate proteins and protein functions from vitreous samples. While this approach continues to provide important new information, especially for low-abundance proteins, emerging opportunities utilizing omic technologies are rapidly expanding our understanding of the complexity of vitreous fluid. Proteomic approaches have identified specific proteins in vitreous that are associated with diabetic retinopathy, and a limited number of these proteins have been shown to induce functional and structural changes in the retina in animal models that are consistent with diabetic retinopathy. Moreover, recent advances in proteomics and bioinformatics are creating opportunities to characterize biological processes that may contribute to diabetic retinopathy and identify biomarkers that further characterize differences in disease progression and responses to therapeutic interventions among patients with seemingly similar disease characteristics. While vitreous proteomics holds exciting potential for expanding understanding of the molecular mechanisms and complexities of diabetic retinopathies, these studies will require methods to integrate the rapidly expanding volume of proteomic data with basic science and clinical aspects of vitreous biology and diabetic retinopathy.
VITREOUS ANATOMY
The vitreous is an optically transparent gel-like fluid that provides both structural and biochemical functions in ocular physiology. The gel-like composition of the vitreous is derived mainly from a hydrated network of fibular macromolecules, including glycosaminoglycans (GAG), proteoglycans, and collagen fibrils. Within this fluid and lattice of macromolecules there is a metabolically active and dynamic biochemical milieu. Soluble proteins can diffuse between the vitreous and retinal interstitial fluid across of the inner limiting membrane (ILM), suggesting that the vitreous may contain information derived from retinal disorders, and proteins in the vitreous can feedback to influence retinal functions and pathologies.
The normal adult vitreous is largely acellular and organized with collagen fibrils oriented along an anterior to posterior axis [2]. The interface between the vitreous and retina involves the posterior vitreous cortex and ILM, which mediate regions of vitreoretinal adhesion. The concentrations of collagen isoforms, including types II, V, IX, and XI, are higher in the vitreous cortex compared to central vitreous [3]. Intravitreal localization of other major component molecules, such as hyaluronan, also varies according to their anatomical distribution within the vitreous. These extracellular matrix (ECM) molecules provide a scaffold that binds ions, water, and soluble proteins, which can influence diffusion within the vitreous compartment. The organization of ECM molecules within the vitreous suggests the possibility that soluble proteins that bind to ECM may also be spatially organized or heterogeneously distributed within this compartment.
The vitreous often undergoes a liquefaction process during aging, which alters the biochemical and anatomical heterogeneity of this structure and can alter oxygen consumption and gradients [4]. Liquefaction of vitreous together with the age-related weakening of adhesion between the vitreous cortex and ILM contributes to vitreoretinal disorders, including rhegmatogenous retinal detachment (RRD) [2, 5]. Changes in vitreous ECM, liquefaction of vitreous during aging, and effects of vitreoretinal traction
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could influence the diffusion, retention, and localization of proteins in the vitreous. Thus, the composition of vitreous samples collected from control subjects and subject patients with diabetic retinopathies is likely influenced by coexisting vitreous disorders.
A CANDIDATE APPROACH
Studies of vitreous during the 1970s and 1980s revealed a number of proteins and biochemical activities within this fluid. These early studies of vitreous identified ironbinding proteins, including transferrin [6], which were suggested to provide a protective role for the retina against the detrimental effects of iron, resultant of vitreous hemorrhage [7, 8]. The vitreous was also shown to contain fibrinolytic activity and complement components, which were implicated as clearance mechanisms for hemorrhage and infection [9, 10]. Further early investigations of vitreous activities identified growth factors, potential regulators of growth factor action, and proteins involved in remodeling [11–15]. These findings, and others, revealed that proteins within the vitreous provide a plethora of biochemical functions in ocular physiology. Moreover, this early work suggested that vitreous may not only contain protein involved in the maintenance of normal ocular physiology but may also contain factors that contribute to retinal diseases, including diabetic retinopathy [16, 17].
A series of reports in 1994 revealed increased abundance of vascular endothelial growth factor (VEGF) in vitreous during ocular neovascularization, experimentally induced retinal ischemia, and PDR [18–20]. Subsequent reports demonstrated that intravitreal injection of VEGF induces retinal vascular permeability (RVP) [21], intravitreal VEGF levels are elevated in DME [22], and inhibition of the VEGF pathway ameliorates DME [23, 24]. These findings have revealed that the vitreous, at least in a subgroup of patients with diabetic retinopathy, contains a key mediator of PDR and DME, namely VEGF.
Over the past 2–3 decades, multiple studies have utilized similar candidate molecule approaches to further characterize changes in proteins, including a variety of chemokines, hormones, growth factors, inflammatory molecules, as well as angiogenic and anti-angiogenic factors, in vitreous from patients with diabetic retinopathy. Funatsu et al. reported that in DME VEGF levels in vitreous correlate with elevated levels of intercellular adhesion molecule-1 (ICAM-1), interleukin (IL)-6, and monocyte chemotactic protein-1 (MCP-1) [25], suggesting a link between VEGF and inflammation. Moreover, elevated levels of these factors correlated with increased RVP and retinal thickness [22, 25]. Yoshimura et al. has shown that IL-6, IL-8, and MCP-1 are elevated in vitreous from PDR and DME compared with nondiabetic (NDM) controls [26], and increases in levels of these inflammatory factors correlated with elevated VEGF levels in vitreous. Platelet-derived growth factor (PDGF)-AA, PDGF-AB, PDGF-BB isoform levels were shown to be elevated in vitreous from subjects with PDR, and increasing concentration of these PDGF isoforms was also shown to correlate with VEGF levels [27]. Moreover, changes in intravitreal levels of insulin-like growth factor-I (IGF-I) and IGF-binding proteins in people with diabetic retinopathy have also been reported [28]. This growing body of work has provided insights into the complexity and heterogeneity of potential hormonal, growth factor, and cytokine influences of the vitreous on diabetic retinopathy. While these finding suggest a variety of protein and pathways that may