Extracellular signalling proteins interact in networks rather than in isolation. In this context we investigated vitreous protein levels, including placental growth factor (PlGF), angiopoietin-2 (ANG2) and vascular endothelial growth factor (VEGF), in patients with proliferative diabetic retinopathy (PDR) with variable disease severities, and after anti-VEGF pre-treatment. Vitreous samples of 112 consecutive patients undergoing vitrectomy for PDR and of 52 non-diabetic patients with macular holes as controls were studied. A subset of the PDR patients were treated with either aflibercept (AFB, n = 25) or bevacizumab (BVZ)/ranibizumab (RZB) (n = 13), before surgery. Antibody-based analysis of 35 proteins (growth factors and cytokines) showed a significant increase in expression levels of 27 proteins in PDR patients as compared to controls. In network analysis of co-regulated proteins, a strong correlation in expression levels between VEGF, PlGF, MCP1 and ANG2 was found, mostly clustered around ANG2. In the AFB treatment group, concentrations of several proteins were decreased, including VEGFR1, whereas interleukin 6 and 8 were increased as compared to untreated PDR patients. The observed differences in vitreous protein levels between the different treatments and untreated PDR patients may underlie differences in clinical outcomes in patients with PDR.