Identification of proteins associated with clinical and pathological features of proliferative diabetic retinopathy in vitreous and fibrovascular membranes

Abstract

PurposeTo identify the protein profiles in vitreous associated with retinal fibrosis, angiogenesis, and neurite formation in epiretinal fibrovascular membranes (FVMs) in patients with proliferative diabetic retinopathy (PDR).MethodsVitreous samples of 5 non-diabetic control patients with vitreous debris and 7 patients with PDR membranes were screened for 507 preselected proteins using the semi-quantitative RayBio® L-series 507 antibody array. From this array, 60 proteins were selected for a custom quantitative antibody array (Raybiotech, Human Quantibody® array), analyzing 7 control patients, 8 PDR patients with FVMs, and 5 PDR patients without FVMs. Additionally, mRNA levels of proteins of interest were measured in 10 PDR membranes and 11 idiopathic membranes and in retinal tissues and cells to identify possible sources of protein production.ResultsOf the 507 proteins screened, 21 were found to be significantly elevated in PDR patients, including neurogenic and angiogenic factors such as neuregulin 1 (NRG1), nerve growth factor receptor (NGFR), placental growth factor (PlGF) and platelet derived growth factor (PDGF). Angiopoietin-2 (Ang2) concentrations were strongly correlated to the degree of fibrosis and the presence of FVMs in patients with PDR. Protein correlation analysis showed PDGF to be extensively co-regulated with other proteins, including thrombospondin-1 and Ang2. mRNA levels of glial-derived and brain/derived neurotrophic factor (GDNF and BDNF) were elevated in PDR membranes. These results were validated in a second study of 52 vitreous samples of 32 PDR patients and 20 control patients.ConclusionsThis exploratory study reveals protein networks that potentially contribute to neurite outgrowth, angiogenesis and fibrosis in the formation of fibrovascular membranes in PDR. We identified a possible role of Ang2 in fibrosis and the formation of FVMs, and of the neurotrophic factors NRG1, PDGF and GDNF in neurite growth that occurs in all FVMs in PDR.

Mousetap, a Novel Technique to Collect Uncontaminated Vitreous or Aqueous and Expand Usefulness of Mouse Models

“…Experiments aimed at identifying proteins contributing to pathologic effects in the retina are another useful application for aqueous and vitreous samples. Almost all of the proteins that were substantially higher in the vitreous of mice with OIR versus age-matched controls have been previously demonstrated to be increased in the vitreous of patients with PDR compared with controls, including PAI-1 23 , IGFBPs 24 – 26 , PTX3 27 , PlGFs 28 , 29 , MMPs 5 , 30 , 31 , TIMP-1 32 , SDF-1 33 , and MCP-1 34 , 35 . These molecular correlations support the anatomic correlations of retinal nonperfusion and retinal NV, indicating that the OIR mouse model recapitulates critical aspects of PDR and that vitreous samples obtained from OIR mice are likely to be useful for investigations of the molecular pathogenesis of PDR and other human ischemic retinopathies.…”

Effect of intravitreal conbercept treatment on the expression of Long Noncoding RNAs and mRNAs in Proliferative Diabetic Retinopathy Patients

“…The degree of haemorrhage, degree of fibrosis, activity of neovascularization were recorded using a standardized form (Smith & Steel ; Klaassen et al. ). Surgeries were routine 25‐gauge PPV, and the preretinal membranes were peeled from the retinal surface using microsurgical forceps.…”

microRNA Expression Profile in the Vitreous of Proliferative Diabetic Retinopathy Patients and Differences from Patients Treated with Anti-VEGF Therapy

“…29 Predicted targets were extracted from miRWalk 3.0. 30 To limit the number of validated and predicted targets, we focused on genes with elevated protein levels in the vitreous of PDR patients according to Klaassen et al 31…”

“…To estimate possible functions of hsa-miR-20a-5p, hsa-miR-23b-3p, hsa-miR-142-3p, hsa-miR-185-5p, hsa-miR-326, and hsa-miR-362-5p, we analyzed validated and predicted targets of these miRNAs. Within these targets, we focused on proteins that are differently expressed in the vitreous of PDR patients 31 and are associated with DR.…”

“…All six miRNAs are predicted to target proteins that are elevated in DR, 31 such as bone morphogenetic protein 2 or angiopoietin 1 (Table 2), or are associated with angiogenesis and wound healing responses, major processes involved in PDR (VEGF, angiopoietin-2, or connective tissue growth factor [CTGF]). VEGF-A is a confirmed target protein for hsa-miR-20a-5p and hsa-miR-185-5p.…”