Study On Vitreous Proteomic Analysis And Biomarker Discovery Of Proliferative Vitreoretinopathy

Purpose: Proliferative vitreoretinopathy (PVR) is the most common cause of anatomic failure in retinal detachment surgery. To understand the molecular mechanisms, vitreous proteomes of rhegmatogenous retinal detachment patients with PVR were investigated by proteomic strategies. The purpose of the study is to separate and identify the vitreous proteome of PVR and donor eyes in order to fish for the serum biomarker which can evaluate the severity of PVR preoperatively and prognosis postoperatively.Methods: (1) To separate and identify the vitreous proteome of PVR by two-dimensional-liquid chromatography coupled with tandem mass spectrometry (2D-LC-MS/MS): Vitreous samples of moderate PVR (grade B), and severe PVR (grade C or D) were aspirated during pars plana vitrectomy (PPV) before infusion and the serum of those patients were collected preoperatively. The normal vitreous from donor eyes and normal serum from healthy humans was as the control. The vitreous samples in the same group were mixed at same volume before the experiment. Cold acetone was added into the vitreous samples overnight. The samples were lysed in Tris base buffer, and then were digested into peptides using sequencing grade trypsin overnight at 37°C. The protein mixtures from the samples were digested and then fractionated into 10 subgroups by strong cation exchange (SCX) chromatography. The peptide mixtures of each SCX fraction were then loaded onto a reverse phase (RP) trap column for desalting. A spray voltage of 2500V was applied to a PicoTip nanospray emitter (New Objective) to give a steady spray. The MS/MS spectras were searched against the human protein databases using MASCOT. (2)The differential vitreous proteome between PVR and donor by two-dimensional gel electrophoresis coupled with matrix assisted laser desorption / ionization - time of flight - mass spectrometry (2-DE-MALDI-TOF-MS) : The samples were same as the first part. The vitreous samples in the same group were mixed at same volume before the experiment. Cold acetone was added into the vitreous samples overnight. First dimension isoelectric focusing (IEF) was performed with the Pharmacia IPGphor in solvent B using 18 cm non-linear pH 3±10 immobilized pH gradient (IPG) strips. IPG strips were rehydrated with sample then IEF was performed at 100 V for 30 min, linear increase to 3500 V for 4 hr, and then held at 8000 V for 4 hr. The second dimension 12% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) was performed at 30 mA for 6 hr after the IPG strips were equilibrated. After silver staining, the gels were analyzed by the 2-DE gel analysis software. The matched spots of which exhibited at least two-fold difference in average optical density (OD) % were excised and tryptic digested in-gel. The pepetides were analyzed by MALDI-TOF-MS and the MS/MS spectras were searched against the human protein databases using MASCOT. (3) To verify kininogen 1 in the vitreous and serum samples. The vitreous of the donor and PVRB, C, D and the corresponding serum were same as the first part. Furthermore, the vitreous of PVR A and the corresponding serum preoperatively was collected. The serum samples from the patients which silicon oil were tamponaded in PPV (group SO) or were uncured after scleral buckling surgery (group SBS) were collected. All the vitreous and serum samples were performed western blotting analysis (WB) and enzyme linked immunosorbent assay (ELISA). (4) To verify insulin-like growth factor binding protein (IGFBP-6)in the vitreous and serum samples. The samples were same as the third part. To verify IGFBP-6, all the vitreous and serum samples were performed WB and ELISA.Results: (1) In the current study, 129, 97 and 137 proteins were identified in vitreous of donor, moderate and severe PVR, including 35 overlap proteins and 24 PVR special proteins. In PVR vitreous samples, complement components, serine proteinase inhibitors, and extracellular proteins were up-regulated or increased, while normal cytoskeleton and enzymes involved in glycolysis, such as pyruvate kinase and enolases, were down-regulated or disappeared. Among the PVR special proteins, kininogen 1 was one of the relatively high abundance proteins of which more peptides could be identified; IGFBP-6 was involved in signal transduction. (2) There were 47, 184 and 336 protein spots were analyzed from the 2-DE gels of donor, moderate PVR and severe PVR. Among 13 the significantly difference spots, 7 proteins were successfully identified. Enolase 2 was the special protein in donor vitreous; transthyretin monomer and retinol-binding protein (RBP) chain B were special proteins in severe PVR vitreous; prostaglandin D synthase and RBP3 precursor were the overlap proteins,which were upregulated in moderate PVR but downregulated in severe PVR; albumin and transferrin were upregulated associated with the severity of PVR. (3) WB outcomes presented that kininogen 1 was detected in both vitreous and the corresponding serum of PVR patients but was not detected in either donor vitreous or normal serum. Kininogen 1 expressed highly in the vitreous and serum of PVR C and D than those in PVRB. Kininogen 1 could be detected in 100% of vitreous and serum samples. The ELISA outcomes indicated that the concentration of kininogen 1 in vitreous and serum samples of severe PVR was significantly higher than in moderate PVR(P<0.05). Kininogen 1 was reduced significantly at 6 month postoperatively(P<0.05), but was still higher than normal (P<0.05). However, kininogen 1 in group SBS was significantly higher than in group SO and normal control (P<0.01), and was still higher than in 6 month postoperative followed-up group(P<0.05). (4) WB outcomes presented that IGFBP-6 was detected in both vitreous and the corresponding serum of PVR patients but was not detected in either donor vitreous or normal serum. The IGFBP-6 expressed highly in the vitreous and serum of PVR C and D than those in PVRB. IGFBP-6 could be detected in 91.7% of vitreous and serum samples except for 2 samples of PVRB. The ELISA outcomes indicated that the concentration of IGFBP-6 in vitreous and serum samples of severe PVR was significantly higher than in moderate PVR(P<0.05). IGFBP-6 was reduced significantly at 6 month postoperatively(P<0.05), and no significant difference was found comparing with the normal and group SO(P>0.05). However, IGFBP-6 in group SBS was significantly higher than in normal, group SO and postoperative group(P<0.05).Conclusions: (1) Out current proteome study presented that PVR was a complicated pathology process with great amount of proteins newly produced, serum proteins invaded in and normal proteins reduced. These outcomes indicated that destruction of blood-retinal barrier, metabolism dysfunction, immune reactions, and cytoskeleton remolding were all involved in the process of PVR. (2) The combination of 2D-LC-MS/MS and 2-DE-MALDI-TOF-MS could be complemented to display the integrated differential proteome. (3) Kininogen 1 and IGFBP-6 may be candidate serum biomarkers of PVR, which can evaluate the severity of PVR and preoperatively and prognosis postoperatively. Further investigations into these special proteins will provide additional targets for treatment or prevention of PVR. Our study provided exciting future in clinical practice...