Besides age-related changes in the proteome, retinal vein occlusion (RVO) is accompanied by specific alterations in the matrix and hemostasis-associated proteins. The pivotal role of monitored changes is impaired blood outflow, so the retina suffers ischemic conditions upstream of the occlusion 15. The ongoing anatomical changes induce an increase in VEGF and favor to drive the inflammatory condition 16. Recent proteomic analysis of vitreous humor samples from RVO patients showed credible up-regulation of pro-inflammatory and immune-related proteins: C3 complement factor, CLU, VTN, and IGLL5 17. Although this small cohort of proteins is of interest, it only indicates common undergoing changes and cannot be used as markers. Animal model of laser-induced BRVO uncovered several interesting alarming proteins that were not reported previously as RVO-associated, but most of them are matrix-related (including laminin subunits, integrin, and actinin isoforms). Based on this evidence, the authors concluded that RVO is tightly associated with the matrix remodeling process as well as integrin and focal adhesion signaling 18.
The study of animal laser-induced RVO revealed, that it could be distinguished from the control samples based on the proteomic data. The main input into discrimination made pro-inflammatory proteins, of which S100A12 was the most profoundly increased 8. Our data also suggests that ischemic conditions in RVO patients might be associated with inflammation and mediated by immune-related proteins since S100A family proteins and B2M (β2-microglobulin) (Table 1) are significantly up-regulated in the RVO patients. These proteins construct a separate cluster on the protein-protein interaction map (S100A6, S100A8, and S100A9; Figure 4) and are tightly joined with matrix-related (ACTB) and metal-binding proteins (TF). Previous comparative studies of CRVO and non-ischemic ocular disease patients revealed that pro-inflammatory proteins level directly correlates with the severity of retinal ischemia 19. If S100A6 is a prolactin receptor-associated protein mediating glucocorticoids secretion, while S100A9 and S100A8 both are regulators of cells migration acting in the defending mechanisms against inflammation, oxidative stress, and innate immune stimulation, one may conclude that these proteins indicate local inflammation as a consequence of CRVO ischemic condition.
The S100 family proteins were rigorously revied in the context of eye disease as promising markers patterns with a high predictive potency and disease monitoring value 20. These proteins have been repeatedly screened in the tear and showed an ability to discriminate between dry eye disease and CRVO due to controversial regulation (upregulated in CRVO) 8,21,22. These secreted proteins are typically well correlated with a significantly suppressed MHC-I peptide representation B2M protein (Table 1) and extracellular matrix proteins abundantly observed in our study, including PRR4, ACTB, MSLN, and CST3 (Table 1) indicating active immune response regarding chemokines activation. Interestingly, but it should be notified, that MSLN (mesothelin) has not been ever reported in tear proteome at either eye-related disease. The protein is generally mentioned as a target in the chemotherapy of different oncophenotypes, and mostly regarding solid tumors 23.
The finding of MSLN in the tear was unexpected, however, if consider the beguiling idea of the increased level of extracellular matrix compounds as a biomarker for CRVO and glaucoma 24–26, it seems possible the presence of MSLN due to its importance in cell adhesion. In consistence with our observation, ischemic condition at CRVO induce the expression of ocular-specific pro-fibrotic TGF-β2 factor, that sufficiently promotes the growth of extracellular matrix 26,27. Therefore, MSLN was established among up-regulated proteins in this study (Table 1). However, it should be admitted the matrix proteins are highly sensitive, so their alteration is generally a consequence of oxidative stress and inflammation, whereas the primary sign is depicted in hemostasis and mitochondria maintenance dysregulation. It has been reported, that patients with glaucoma and CRVO are characterized by irregular levels of GPX, SOD, and MDA, and typically present a higher level of these proteins even after surgery 28,29.
The association of oxidative stress and macular degeneration with damaging and dysfunction of mitochondrial 30. Moreover, with aging mitochondria become progressively more incomplete, thus CRVO can be considered as an age-related disease with a pivotal role in increasing ROS output 31. In this study, the oxidative stress consequence is reflected in the decrease of mitochondria and mitochondria inner membrane organization processes (Figure 3), possibly indicating the prevalence of apoptosis and cell-damaging. In oppose, processes related to cell response of topologically incorrect proteins and chaperon-cofactor proteins refolding and de novo proteins folding are substantially up-regulated in the proteome of patients with CRVO (Figure 3) along with chaperon proteins (Table 1).
While all retinal cells rely on ATP as a fuel source and the photoreceptors are the largest consumers requiring regular mitochondria functioning, hypothesized, that such cautioning processes (Figure 3) underline consequences of the impaired retinal hemostasis and accompanied tissue-damaging caused by the increased pro-inflammatory activity. Furthermore, stimulated mitochondrial oxidative phosphorylation contributes significantly to the increased ATP and, consequently, ROS generation and may cause protein folding and refolding processes in the endoplasmic reticulum 32 and stimulates the production of inflammatory cytokines 33. Notwithstanding, only a few reports can be found regarding the relation of oxidative stress with CRVO 28, but much more reports review the relation with diabetic retinopathy 34–36.
If considering oxidative stress as the paramount background event of CRVO, it is essential to unfurl the role of ROS scavengers and the related transport system as a possible victim of the boosted ROS generation and cytokines production. We found the local increase of transthyretin (Table1) also known as transthyretin (TTR) in the proteome of CRVO patients with a 0.88 frequency. The protein is mainly synthesized in the liver and is also found to be expressed in retinal pigment epithelial cells 37. Apart from the pivotal role of TTR in the transport of thyroxine to the brain, circulating TTR also carries a retinol-binding protein (RBP) transporting and stabilizing retinol as an essential antioxidant agent. Due to high toxic potency, retinol is primarily stored in form of fatty acid esters. Vitamin A can efficiently neutralize thiol-radicals and stabilizes peroxide radicals being an important epigenetic factor preventing and reducing the risk of oxidative stress and inflammatory-related diseases 38,39. The deficiency of TTR may aggravate the risk of oxidative stress and enhance local impairment of circulation and angiogenic processes, which is particularly established for cardiovascular and nondegenerative diseases 40. On the other side of this matter, excessive circulating TTR provides a favorable environment for the transthyretin amyloidosis being involved in numerous ophthalmological pathologies 37.
Despite most of the studies intend to assemble proteins in a panel of putative biomarkers, we are reluctant to build the decision-making pattern of CRVO biomarkers. Instead, we focused on the five most significantly altered proteins credibly associated with the pathogenesis of CRVO and capable of indicating the ongoing inflammation caused by the ischemic condition. We submitted S100A6, S100A8, S100A9, MSLN, and B2M proteins to estimate the area under the curve (Figure 5). The obtained measures of ROC for each protein satisfies specificity and sensitivity expectations. Although some of these proteins are repeatedly mentioned in the context of CRVO severity and onset, their combination might be a more proper item for consideration to figure out the very beginning of CRVO and to monitor the bases of ocular diseases treatment.