Identification of Potential Biomarkers for Rhegmatogenous Retinal Detachment Associated with Choroidal Detachment by Vitreous iTRAQ-Based Proteomic Profiling

Rhegmatogenous retinal detachment associated with choroidal detachment (RRDCD) is a complicated and serious type of rhegmatogenous retinal detachment (RRD). In this study, we identified differentially expressed proteins in the vitreous humors of RRDCD and RRD using isobaric tags for relative and absolute quantitation (iTRAQ) combined with nano-liquid chromatography-electrospray ion trap-mass spectrometry-mass spectrometry (nano-LC-ESI-MS/MS) and bioinformatic analysis. Our result shows that 103 differentially expressed proteins, including 54 up-regulated and 49 down-regulated proteins were identified in RRDCD. Gene ontology (GO) analysis suggested that most of the differentially expressed proteins were extracellular．The Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis suggested that proteins related to complement and coagulation cascades were significantly enriched. iTRAQ-based proteomic profiling reveals that complement and coagulation cascades and inflammation may play important roles in the pathogenesis of RRDCD. This study may provide novel insights into the pathogenesis of RRDCD and offer potential opportunities for the diagnosis and treatment of RRDCD.


Introduction
Rhegmatogenous retinal detachment associated with choroidal detachment (RRDCD) is a complicated and serious type of rhegmatogenous retinal detachment (RRD). Clinical symptoms of RRDCD include severe uveitis, ocular hypotony, deep anterior chamber, concentric folding, and iridophakodonesis [1][2][3]. RRDCD progresses rapidly and has a poor prognosis because it quickly proceeds to severe proliferative vitreoretinopathy (PVR). The reported incidence of RRDCD in primary RRD is 1.5% to 18.1% in China and 2.0% to 4.5% in Western countries [2,4,5]. RRDCD usually occurs in patients with high myopia, pseudophakia, aphakia, and increased age [5,6]. A recent study has shown that retinal detachment associated with macular hole predisposes one to RRDCD [7]. Visual prognosis after pars planavitrectomy, the conventional treatment for RRDCD, is still unfavorable. The high incidence of PVR after surgery may contribute to the low reattachment rate [8]. To date, most studies have mainly focused on clinical problems. However, the etiology and pathogenesis of RRDCD remain elusive. It is generally believed that hypotony is the initial step of RRDCD [5]. Another hypothesis [3] proposes that intraocular inflammation secondary to retinal detachment plays a pivotal role in the development of choroidal detachment. Given the severe uveitis signs of RRDCD, better prognosis obtained through preoperative application of steroids, as well as our previous study showing that inflammation may participate in the process of RRDCD [9,10], we assume that inflammation has a key role in the occurrence and development of RRDCD.
Proteomics is a post-genomic discipline that studies the proteins of a proteome by various techniques. Since biological activity is executed directly by proteins, studying proteomics in disease or different stages of diseases may shed light on etiopathogenesis and deepen our understanding of diseases, and further help develop better treatment strategies for various diseases. Isobaric tags for relative and absolute quantitation (iTRAQ) is a new technique for protein separation consisting of a unique mass reporter, a mass balancer, and an amine-reactive group [11]. Several features of iTRAQ, such as high through-put, wide range of separation, high accuracy, and repeatability, have led to its widespread use in proteomics.
To date, few studies have studied human vitreous samples from RRDCD patients by proteomic profiling. In this study, we investigated protein profiles of vitreous humor and identified protein biomarkers by iTRAQ combined with nano-liquid chromatography-electrospray ion trap-mass spectrometry-mass spectrometry (nano-LC-ESI-MS/MS) in RRDCD. The objective of this study was to discover differentially expressed proteins and analyze biological processes to improve our understanding of the etiopathogenesis and the poor prognosis of RRDCD. Table 1 shows the demographic and clinical data of the patients enrolled in the study, including 8 RRDCD and 8 RRD patients. There were no significant differences in sex, age, and duration of detachment in the two groups (p > 0.05). The RRDCD patients had significantly worse PVR compared to the RRD patients (p < 0.001). In addition, the intraocular pressure (IOP) was significantly lower in RRDCD than in RRD patients (p < 0.005).   Red represents up-regulated protein expression of RRD compared to RRDCD, whereas green represents down-regulated protein expression of RRD compared to RRDCD.
GO enrichment analysis of the differentially expressed proteins showed that the GO terms of most differentially expressed proteins were enriched in extracellular space (p = 9.29 × 10 −7 ), followed by biological regulation (p = 0.001) and regulation of biological process (p = 0.0003, Figure 3).

Kyoto Encyclopedia of Genes and Genomes Analysis
Using the Kyoto encyclopedia of genes and genomes (KEGG) pathway database, the differentially expressed proteins were classified into 65 pathways, the top 21 of which are shown in Figure 4. The complement and coagulation pathway was the top pathway, followed by the systemic lupus erythematosus (SLE) pathway. KEGG analysis showed that the significantly enriched pathways included the complement and coagulation and SLE pathways ( Figure 5). The KEGG maps are shown in Figure 6.

Protein Verification by Western Blotting Analysis
C3, prothrombin, kininogen-1, and IgG heavy chain were randomly chosen and detected in all RRD (n = 8) and RRDCD (n = 8) vitreous samples by Western analysis. Changes in protein abundance shown by Western analysis and quantification of the proteins were highly consistent with the proteomic data of patients ( Figure 7).

Protein Verification by Western Blotting Analysis
C3, prothrombin, kininogen-1, and IgG heavy chain were randomly chosen and detected in all RRD (n = 8) and RRDCD (n = 8) vitreous samples by Western analysis. Changes in protein abundance shown by Western analysis and quantification of the proteins were highly consistent with the proteomic data of patients (Figure 7).

Discussion
This study investigated the proteomic profiles of RRDCD vitreous humor using the iTRAQ and nano-LC-ESI-MS/MS techniques. We identified 103 differentially expressed proteins, including 54 up-regulated and 49 down-regulated proteins, in the RRDCD samples compared to the RRD samples. We randomly selected four proteins for validation by Western blot analysis. The changes in protein abundance were highly consistent with the proteomic data.
According to GO annotation analysis, the differentially expressed proteins in the RRDCD samples were primarily assigned to biological regulation, binding, and extracellular region when analyzed for BP, MF and CC, respectively. GO enrichment analysis showed that most of the

Discussion
This study investigated the proteomic profiles of RRDCD vitreous humor using the iTRAQ and nano-LC-ESI-MS/MS techniques. We identified 103 differentially expressed proteins, including 54 up-regulated and 49 down-regulated proteins, in the RRDCD samples compared to the RRD samples. We randomly selected four proteins for validation by Western blot analysis. The changes in protein abundance were highly consistent with the proteomic data.
According to GO annotation analysis, the differentially expressed proteins in the RRDCD samples were primarily assigned to biological regulation, binding, and extracellular region when analyzed for BP, MF and CC, respectively. GO enrichment analysis showed that most of the differentially proteins were enriched in the extracellular space. These proteins may be secreted from the neighboring tissues or derived from the broken blood-retinal barrier (BRB). The complement, coagulation and SLE pathways were significantly enriched in KEGG pathway analysis.
Most up-regulated proteins in the vitreous humor of the RRDCD group, compared to those in the RRD group, were plasma proteins. This might be caused by the leakage from the suprachoroidal space or retinal vessels into the vitreous. These proteins might not be inductive factors for RRDCD. It is possible that the level of protein concentration is useful for diagnosis or predictions of RRDCD.
This study identified highly abundant proteins associated with inflammation in RRDCD, such as α-1-antitrypsin, immunoglobulin chains and complement proteins (C1r, C3, C6 and C8). α-1-antitrypsin is a member of the serpin superfamily. Its typical function is inhibiting serine protease like neutrophil elastase, proteinase 3, trypsin, kallikreins and so on [13]. α-1-antitrypsin is an acute-phase response prtein and involves in reducing production of inflammatory mediators, blocking inflammatory cells to modulates inflammatory responses [14]. The previous studies show that complement components and immunoglobulins existed in epiretinal membranes of PVR [15,16]. The immunoglobulin heavy and light chains were significantly up-regulated in the vitreous of RRDCD, indicating that the immune reaction or BRB breakdown in RRDCD was more serious than in RRD.
The complement system is an important constituent of human innate immunity and a bridge to adaptive immunity [17]. C3a is product of C3 activation, also known as anaphylatoxin. Activation of the G protein coupled receptors (C3aR) by C3a promotes degranulation in mast cells and basophils and release of histamine and other mediators, finally resulting in inflammatory hyperemia and edema [18][19][20]. C3a recruits neutrophils, macrophages and other inflammatory cells and induces vasodilation and inflammatory mediator release [18][19][20]. High expression of C3 in the RRDCD vitreous humor can result in intraocular inflammation and destruction of ocular tissues like retina and vessels. This process may lead to RRDCD pathophysiological processes. C6 and C8 participate in the formation of the membrane attack complex (MAC) C5-C9, and subsequently lead to cell lysis [21]. Cashman and colleagues found that mice injected with C3-expressing adenovirus significantly increased vascular permeability, RPE atrophy, endothelial cell proliferation, and migration [18]. Our results support this previous study suggesting a role of complement in RRDCD. The complement components may cause increased retinal vascular permeability, retinal damage, cell proliferation and inflammation, which in turn to promote RRDCD and contribute to PVR and poor visual prognosis.
In this study, coagulation proteins, such as prothrombin, α-1-antitrypsin, α-2-antiplasmin and plasminogen, were significantly up-regulated in the RRDCD vitreous humor. This may be due to the penetration of plasma proteins due to broken BRB and dysfunction of retinal vessels. Blood coagulation is a process in which successive activation of coagulation factors triggers a chain reaction, resulting in fibrin clot formation. The activated coagulation cascade has a positive feedback effect [22,23]. Additionally, there is a reciprocal relationship between coagulation and inflammation [24,25]. Thrombin, which is activated from prothrombin and was identified in this study, is a serine protease that mediates a strong inflammatory response [25]. Up-regulated coagulation proteins may further aggravate ocular inflammation. The coagulation cascade also has a reciprocal relationship with the complement system. The dysregulation of one system can escalate the activation of both. A previous study revealed that plasminogen mutually and prominently activates and coexists with complement in many chronic inflammation diseases [26]. Coagulation cascade components have been found in vitreous humor by some proteomic studies for other vitreoretinal diseases [27,28], but significant enrichment in RRDCD has not been reported. The significantly enriched coagulation components in RRDCD compared to RRD indicate that increased coagulation activity plays a role in the development of RRDCD. This study showed that kininogen-1 and plasma kallikrein B, the components of the kallikrein-kinin system, were up-regulated in the vitreous humor of RRDCD compared to RRD.
The kallikrein-kinin system plays important roles in many physiological and pathological processes such as regulation of vasomotor tone, blood coagulation, fibrinolysis process, inflammation, and electrolyte balance [29][30][31][32]. Plasma kallikrein and high molecular weight kininogen (HMWK) circulate as a complex that binds to plasma membrane of the endothelial cells, therefore, we can assume that the complex also exists in retinal vessels. A previous study has reported that high levels of bradykinin B1 and B2 receptors are expressed in all of the layers of human retina and the vascular endothelial cells [33]. This suggests that the plasma kallikrein/kininogen might participate in the regulation of retinal vessel functions. It has been reported that intraocular hemorrhage increases retinal vascular permeability and leukocyte stasis induced by plasma kallikrein [34]. Another study has shown that intravitreal injection of plasma kallikrein in rats increases retinal vascular permeability [35]. The components of the kinin-kallikrein system probably cross the BRB into the vitreous fluid and enhance vascular permeability. Additionally, plasma kallikrein/kininogen might interact with coagulation components and inflammatory factors, finally promoting PVR and RRDCD disease progression.
Some other proteins, like apolipoprotein, serum albumin, hemopexin, and retinol binding protein 4 (RBP4), were up-regulated in the vitreous of RRDCD. These proteins are important for maintaining cell activities and transport functions. Recently, a few studies indicated that increased RBP4 in blood may promote retinal dysfunction [34,36,37].
Most down-regulated proteins in the vitreous humor of the RRDCD group compared to those in the RRD group were development related proteins. This may indicate that the pathogenesis of RRDCD is more severe and complicated compared to RRD. Prostaglandin-H2 D-isomerase (PTGDS) catalyzes change of prostaglandin H2 to prostaglandin D2 in the arachidonic acid (AA) metabolic pathway. In our study, PTGDS was down-regulated in the vitreous of RRDCD, consistent with the results in our previous metabolomics study of down-regulated AA in the vitreous of RRDCD patients [10].
Although proteomic techniques were already developed and can obtain accurate data to provide additional insights into the development of RRDCD, there were several limitations in this study. First, the number of vitreous samples was relatively small. RRDCD is a disease with low prevalence. In our department, only 13 samples of RRDCD were collected between September 2014 and September 2015, after RRDCD patients administered with preoperative steroids and patients with recurrent RRDCD, secondary RRDCD and comorbid diseases were excluded. However, there was statistically significant difference between the two groups in our study, and the results of WB were consistent with the proteomic data, suggesting that the results are credible; Second, due to the limited number of reliable and constantly updated reference databases for protein identification, our interpretation of the results was limited; Lastly, it is difficult to know what to make of protein interaction networks in a fluid like vitreous humor. Our data offer another perspective to understand the function of retina and choroid in process of RRDCD. The results and conclusions of this study still need to be improved by expanding the samples' size and verified by blood samples in the future research.

Patients
Eight primary RRD and 8 primary RRDCD patients, undergoing pars plana vitrectomy between September 2014 and September 2015 at Nanjing Medical University Affiliated Wuxi Second Hospital (Wuxi, China) were enrolled in this study. The exclusion criteria included diabetic retinopathy, intraocular inflammation, systemic disease such as hypertension, diabetic, previous intraocular surgery and intravitreal injection with steroid or anti-vascular endothelial growth factor. All subjects received comprehensive ophthalmic examination before operation by two experienced doctors. The grade of Proliferative vitreoretinopathy (PVR) was recorded on the basis of the classification of the Retina Society Terminology Committee [38]. The symptom duration was recorded in days and the extents of retinal detachment were recorded in quadrants.

Ethics Statement
The study was followed the principles of Helsinki Declaration and was approved by the ethics committee of Nanjing Medical University affiliated Wuxi NO.2 Hospital (Wuxi, China. Identification code: WXEYLL256. 29 August 2014). All subjects in this study signed a copy of informed consent prior to participation.

Sample Preparation
Approximately 0.5-1.0 mL vitreous samples were collected using a 5 mL sterile syringe when the 25-gauge trocar was inserted into the vitreous cavity before active infusion. Undiluted vitreous fluid was transferred into 1.5 mL Eppendorf Tubes immediately and centrifuged at 9398× g for 10 min at 4 • C. The supernatants were aliquoted and stored at −80 • C until analysis. The samples were thawed and mixed with 800 µL SDT lysis buffer (4% SDS, 100 mM Tris-HCl, 1 mM DTT, and pH 7.6), followed by sonication. After being incubated in a 95 • C water bath for 15 min, the samples were centrifuged at 14,000× g for 40 min. The protein concentration of the supernatants was determined by Bicinchoninic acid protein assay kit (Beyotime, P0012, Nantong, China).

Protein Digestion and iTRAQ Labeling
Filter aided proteome preparation (FASP) was performed as previously described for proteins digestion [39]. The finally filtrates were collected and the peptides were quantified at 280 nm. According to the manufacturer's instructions, 100 µg samples from each group were labeled by iTRAQ Reagent-4plex multiplex kit (AB SCIEX, Foster City, CA, USA). Proteins from RRDCD group and RRD group were labeled with reagent 114 and reagent 116 respectively. Blank control group were labeled with reagent 117.

Nano-LC-ESI-MS/MS Analysis
The fractions were analyzed using Q Exactive MS equipped with Easy nLC (Thermo Finnigan, San Jose, CA, USA). The chromatographic columns were balanced with buffer A (0.1% formic acid). Peptide mixture (2 µg) was loaded via an auto-sampler into a Thermo scientific EASY column (2 cm × 100 µm, 5 µm-C18) and separated by analytical columns (75 µm × 100 mm, 3 µm-C18) at a flow rate of 250 nL/min. The linear gradient was set as 0%-100% buffer B (10 mM KH 2 PO 4 , 500 mM KCl, 25% ACN, and pH 3.0) for 240 min. After separation of nano-HPLC, samples were analyzed by the Q-Exactive mass spectrometer in a positive ion mode. The parent Ion Scan range set as 300-1800 m/z. 10 MS2 scans were collected after every full scan to obtain the mass-to-charge ratio of peptides/peptides fragments.

Protein Identification and Quantification
The raw dates from Q Exactive were submitted to Mascot search software (version 2.2.1; Matrix Science, London, UK) and Proteome Discoverer 1.4 (Thermo Electron, San Jose, CA, USA) for date filtering and proteins identification. Furthered analysis was performed according to false discovery rate (FDR) ≤0.01 using Proteome Discoverer 1.4. Protein identification was supported by at least one unique peptide identified using the Peptide Prophet algorithm. Proteins with fold change >1.2 were retained. Hierarchical clustering analysis was performed by MeV 4.7 software, and Pearson's correlation analysis was used for the distance matrix and the Ward's linkage.

Bioinformatic Analysis
Proteins/peptides sequences were imported into Blast2GO for GO annotation and enrichment [12]. The differentially expressed proteins were then blasted against KEGG GENES to retrieve their KEGG Orthology (KOs) and were mapped to KEGG pathways [40]. Fisher's exact test was carried out to evaluate the enrichment of GO terms and KEGG pathways of differentially expressed proteins against the annotation of all identified proteins.

Conclusions
We performed a proteomic study using iTRAQ-nano-LC-ESI-MS/MS techniques and bioinformatic analyses to identify differentially expressed proteins between RRDCD and RRD. Based on our findings, the proteins of complement and coagulation cascades, the kallikrein-kinin system, and inflammation may play a crucial role in the course of RRDCD. This study provides insights into the development of RRDCD and warrants further studies in the future.   Uniprot accession numbers that can be found on www.uniprot.org; Coverage: The percentage of the protein sequence covered by identified peptides. Unique Peptides: The number of peptide sequences unique to a protein group. Peptides: The number of distinct peptide sequences in the protein group. Fold change:

Appendix A
The quantity changes of protein abundance between the two groups.