Quantitative proteomics analysis of vitreous body from type 2 diabetic patients with proliferative diabetic retinopathy

Diabetic retinopathy (DR), a pathological condition in which damage occurs to the retina due to diabetes mellitus, is the most common microvascular complication of diabetes [1]. This disease can be further classified into two types: nonproliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR). Classification of NPDR is based on clinical findings manifested by visible features, including microaneurysms, retinal hemorrhages, intraretinal microvascular abnormalities (IRMA), and venous caliber changes, while PDR is characterized by the hallmark feature of pathologic preretinal neovascularization [2]. With the increasing global prevalence of diabetes, DR is the major cause of vision loss and blindness among working-age adults in developed countries [3]. Current treatments for diabetic eye disease, which include laser photocoagulation, intravitreous injections of anti-VEGF and steroid agents and vitreoretinal surgery, mainly focus on advanced diseases such as PDR [4]. However, because of side effects and individual differences, there is an urgent need for new therapeutic options for PDR [5‐7]. Therefore, a better understanding of the pathological mechanism is required for the development of new treatment options.

Research on PDR is limited because no current model can completely replicate the full pathophysiology of neuronal and vascular changes that occur at each stage of DR [8]. The physiologic and pathologic conditions of the retina are reflected in the protein composition of the vitreous due to their close anatomical and biological relationship, which can be sampled as part of routine surgical procedures [9, 10]. In this regard, vitreous fluid obtained through vitrectomy is currently used to indirectly examine the condition of the retina. Mass spectrometry (MS) has been widely applied to study biomolecules and one rapidly developing field is the global analysis of proteins, proteomics [11]. MS-based proteomics is of great value to study different ocular diseases such as cataracts [12], age-related macular degeneration (AMD) [13, 14] and DR [15, 16]. Despite the great efforts that have been devoted to DR vitreous protein identification [17, 18], we are still far from a comprehensive understanding of the complex molecular pathomechanisms. Quantitative proteomics is an analytical chemistry technique for determining the amount of proteins, which can contribute to better insight into the underlying pathogenesis [19].

In this study, the abundance of vitreous proteins was compared between the PDR and IMH subjects through a label-free quantitative method. We also analyzed protein distribution and then conducted bioinformatic analyses, which included protein function, related disease, gene ontology (GO), protein-protein interaction (PPI) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway.

Hitherto, there have been several proteomics studies conducted on DR using serum [23‐25], tear fluid [26‐28], aqueous humor [29] and vitreous [30, 31] from patients. However, only a few have quantitatively analyzed PDR [32]. Loukovaara S et al. analyzed 138 vitreous humor samples from patients with NPDR or PDR which had been the most extensive diabetic vitreous proteome analysis so far [20]. Gao BB et al. studied characterization of the vitreous proteome in diabetes without diabetic retinopathy and PDR [33]. However, these two studies both compared PDR and patients who were in a diabetic condition, and thus could not reveal the comprehensive understanding of the underlying molecular pathogenesis of PDR.

García-Ramírez M et al. compared proteomic analysis on vitreous humor from type 1 diabetic patients with PDR and from non-diabetic patients with IMH using fluorescence-based difference gel electrophoresis (DIGE) [32]. Our study was carried out on type 2 diabetic patients with PDR and on IMH subjects through MS-based label-free quantitative proteomics analysis. In this study, the proteomic analysis of PDR vitreous was optimized in the following ways. First, strict exclusion criteria were applied to protect our result from interference, such as infection, hemorrhage or opacities. Second, our proteomic analysis was quantitative, which could obtain the exact abundance of the proteins detected in the samples so that the difference of protein expression in these two diseases could be quantified. An analysis of protein distribution was carried out so that we could gain a brief understanding of protein content, sample reliability as well as a clear separation of the two disease states. Detailed bioinformatic analysis was conducted, which included protein function, related disease, biological processes, molecular functioning, cellular components and the KEGG pathway. Therefore, many inspirations about the target protein function, biomarkers, physiological and pathological changes and the related pathways were revealed through this study.

To our knowledge, this research was the first quantitative proteomics analysis to study the protein distribution which could obtain a brief understanding of protein content and sample reliability. A clear separation of the protein expression in the two diseases was observed. When only taking significant proteins into account, the separation became more apparent and the difference among the samples in one group became less because confounding factors were reduced. Besides, more proteins in the control group had abnormal abundance and C5 was the most unreliable one, the reason of which could be that IMH was another disease condition which might be the result of a variety of unknown factors. Due to its complexity, we just counted IMH samples as controls but did not analyze the related proteins or statistics. Moreover, although there was a weaker correlation among IMH samples, a wider protein distribution in PDR samples was detected which illustrated that PDR was a complex disease which influenced a great deal of protein expression.

A comprehensive bioinformatics research was carried out which provided us with a better understanding of the pathomechanism of PDR as well as the molecular signaling pathway, thus could be of great value to the development of therapeutic method. Apolipoproteins have been reported involving with the pathology of PDR [34, 35], here we identified a total of 6 apolipoproteins, 5 were the first time to be found related to PDR while the remaining one, apolipoprotein A-IV (APOA4) was found reduced in the PDR patients compared with the macular hole patients [36] which had conflicts with our result. Besides, several researches related to PDR have been carried out on vision related proteins, such as CA1 [37], CAT [38], CFH [39], PON1 [40] and VTN [41]. However, no researches have been conducted on APOF, COL1A1, C2, C4BPA, CFD and CFHR3, FGA. Furthermore, KNG1 has never been studied in the pathology of PDR. It initiates the kallikrein-kinin system which is related to the blood pressure systems and inflammation, the latter one is known to cause dilation of blood vessels and increase vascular permeability.

However, there are some limitations in our study. First, the IMH patients were enrolled as controls. Although IMH has no protopathy, it does not equal to normal condition. Because any pathological state may lead to changes in protein profile, normal eyes are the best options for the control group, whereas it is against the ethic. Second, further validation study like dot blot, western blot, enzyme-linked immunosorbent assays or immunohistochemistry should have been conducted in order to confirm some proteins of particular interest. Furthermore, the role of a protein in pathogenesis could have been tested by injection of purified protein into an animal model or by using a pharmaceutical invention.

In conclusion, we identified and quantified 610 proteins in total, which included 338 significant proteins in our study. Protein distribution analysis demonstrated a clear separation of protein expression in PDR and IMH. The protein function analysis illustrated that immunity and transport related proteins might be associated with PDR. In addition, 12 proteins were identified that were related to vision diseases, some of which have already been studied, but more studies should be carried out to analyze the relation between these proteins and PDR as well as the underlying mechanisms. Meanwhile, GO annotation indicated that the proteins in the vitreous may reflected the physiological and pathological changes in retinal lesion. Also, PPI analysis in our study identified the most significant 8 proteins related to PDR. Furthermore, pathway research indicated the complement and coagulation cascades might be the most important pathway and KNG1 is the key protein in the pathology of PDR.