Novel Possible Protein Targets in Neovascular Age-Related Macular Degeneration: A Pilot Study Experiment

Abstract

Age-related macular degeneration (AMD) is among the world's leading causes of blindness. In its neovascular form (nAMD), around 25% of patients present further anatomical and visual deterioration due to persistence of neovascular activity, despite gold-standard treatment protocols using intravitreal anti-VEGF medications. Thus, to comprehend, the molecular pathways that drive choroidal neoangiogenesis, associated with the vascular endothelial growth factor (VEGF), are important steps to elucidate the mechanistic events underneath the disease development. This is a pilot study, a prospective, translational experiment, in a real-life context aiming to evaluate the protein profiles of the aqueous humor of 15 patients divided into three groups: group 1, composed of patients with nAMD, who demonstrated a good response to anti-VEGF intravitreal injections during follow-up (good responsive); group 2, composed of patients with anti-VEGF-resistant nAMD, who demonstrated choroidal neovascularization activity during follow-up (poor/non-responsive); and group 3, composed of control patients without systemic diseases or signs of retinopathy. For proteomic characterization of the groups, mass spectrometry (label-free LC-MS/MS) was used. A total of 2,336 proteins were identified, of which 185 were distinctly regulated and allowed the differentiation of the clinical conditions analyzed. Among those, 39 proteins, including some novel ones, were analyzed as potential disease effectors through their pathophysiological implications in lipid metabolism, oxidative stress, complement system, inflammatory pathways, and angiogenesis. So, this study suggests the participation of other promising biomarkers in neovascular AMD, in addition to the known VEGF.

Keywords: AMD (age-related macular degeneration); biomarkers; choroidal neo vascularization; mass spectrometry (MS); proteomics; resistance.

Figure 1

Flowchart of patient recruitment and exclusion for aqueous humor proteome analysis. The 15 patients selected were separated into three groups: (A) good response to anti-VEGF therapy (nAMD good responsive); (B) resistance to anti-VEGF therapy (nAMD poor/non-responsive); (C) control patients without systemic diseases or signs of retinopathy.

Figure 2

Patient's OCT. (A) good responsive group; (B) poor/non-responsive group; (C) control group.

Figure 3

Hierarchic cluster analyses (HCA) of global protein profiles. Each line represents a protein and each peak or valley, its abundance per patient. Proteins in (A) have a predominance of positive regulation in cases of good responsive nAMD and poor/non-responsive nAMD with the predominance of negative regulation in controls. In (B), the opposite relationship is observed.

Figure 4

Differentially expressed proteins converge on main pathways by biological processes. The links between proteins represent interactions, according to the pattern of the String program (protein-protein interaction network enriched with functional analysis). ENPP2, ectonucleotide pyrophosphatase/ phosphodiesterase family member 2; KDR, kinase insert domain receptor (A type III receptor tyrosine kinase), isoform CRA_a; IGFBP7, insulin-like growth factor-binding protein 7; IGFBP5, insulin-like growth factor-binding protein 5; CFHR1, complement factor H-related protein 1; C4A, complement C4-A; APOA1, apolipoprotein A-I, isoform CRA_a; VTNC, vitronectin; C3, tetranectin; C8A, complement component C8 alpha-chain; APOA4, apolipoprotein A-IV; RBP4, retinol-binding protein 4; SERPINF1, pigment epithelium-derived factor; LCN1, lipocalin 1 (tear prealbumin), isoform CRA_a; C7, complement component C7; APCP1, amyloid-like protein 1; CRYBB2, beta-crystallin B2; IMPG1, interphotoreceptor matrix proteoglycan 1; CRYBB1, beta-crystallin B1; UCHL1, ubiquitin carboxy-terminal hydrolase; KLKB1, plasma kallikrein; RBP3, retinol-binding protein 3; CAT, cathepsin D; MT1G, metallothionein 1G; SOD3, extracellular superoxide dismutase [Cu-Zn]; ENO1, enolase 1 (alpha), isoform CRA_a.

Figure 5

Multivariate analyses by VIP scores of regulated proteins in aqueous humor of patients with good responsive nAMD, poor/non-responsive nAMD, and control patients (PLS-DA imp. features). Among the 30 regulated proteins with the highest VIP score, 25 have negative regulation in pathological scenarios. The colored boxes on the right indicate the relative intensity of each protein in the respective scenarios.