To assess protein expression differences between AMD patients and controls, identify biomarkers of AMD and identify relevant networks of biological pathways, a comprehensive systematic review and meta-analysis was performed based on mass spectrometry-based proteomics of human intralocular fluids. A total of 161 proteins were identified as altered between the AMD and control groups.
GO analysis and KEGG analysis were performed on all the DEPs. GO analysis showed a connection between AMD and the proteolysis, innate immune response, and negative regulation of endopeptidase activity, which mainly occurred in extracellular exosomes, extracellular regions and the extracellular space. In terms of MF, identical protein binding and calcium ion binding were the most important functions. KEGG showed that the most significant pathway was complement and coagulation cascades. The results of GO and KEGG analyses were generally consistent with the known pathogenic mechanism of AMD.[6]
The meta-analysis on DEPs in AH found upregulated APOA1, C3, LCN1 and downregulated PTGDS, while the meta-analysis on VH showed IGHG1, IGKC, IGLC2, PTGDS and TF to be overexpressed.
PTGDS is downregulated in the AH [23, 25, 26] but upregulated in the VH of AMD patients.[30, 31] PTGDS is an enzyme that converts prostaglandin H2 to prostaglandin D2 and acts as a transport protein for lipophilic substances such as retinoic acid and bilirubin.[33] The relationship between PTGDS and AMD was confirmed by analysis of blood samples.[34] PTGDS is considered a protective factor because it can prevent oxidative stress and apoptosis-related neurodegenerative diseases.[35] The reason for the different trends in PTGDS may be the various degrees of AMD. The retinal pigment epithelium (RPE) is the main source of intraocular PTGDS,[36] and mild AMD patients with normal RPE functions may exert protective effects by increasing the level of PTGDS. When RPE functions are damaged to a certain extent in advanced AMD, the level of PTGDS may decrease.
Further study on significant proteins was conducted through PPI analysis. The results indicated that TF, APOA1 and C3 and LCN1 may be the most important proteins, with the former 3 proteins interacting with each other. Based on the results of KEGG analysis, TF was involved in the HIF-1 signaling pathway. C3 was involved in complement and coagulation cascades and Staphylococcus aureus infection pathways. However, APOA1 and LCN1 were not found in the KEGG pathway results.
TFs are iron binding transport proteins that are responsible for the transport of iron from sites of absorption and heme degradation to those of storage and utilization. Serum transferrin may also have a further role in stimulating cell proliferation.[37, 38] The serum level of transferrin was found to be higher in the AMD group.[39] Transferrin receptor and variability of its gene might also influence AMD risk.[40, 41] Recent studies discovered that hypoxia may aggravate ferroptosis in RPE cells and then affect the pathophysiology of AMD,[42, 43] which provided novel insight into hypoxia, oxidative stress and iron metabolism in AMD pathophysiology. Furthermore, transferrin nonviral gene therapy also showed preliminary effectiveness for the treatment of dry AMD.[44] The above findings indicate that TF may be a promising biomarker of AMD.
APOs are very important in lipid homeostasis by transporting cholesterol and lipids between cells, having a well-established role in the transport and metabolism of lipids and in inflammatory and immune response regulation.[45, 46] APOA1 is one of the major components of high-density lipoprotein (HDL) and is recognized for regulating the plasma levels of free fatty acids, playing an important role in HDL and triglyceride-rich lipoprotein metabolism in the reverse cholesterol transport pathway.[47] Three meta-analyses have shown elevated APOA1 levels in the AH of AMD patients.[23, 25, 26] indicating that APOA1 may be a potential biomarker for AMD.
The complement system is considered to play a central role in AMD pathogenesis, and overactivation of the alternative complement pathway is one of the main drivers of diseases and is related to multiple pathogenic factors of AMD, such as inflammation, oxidative stress and lipid accumulation.[48, 49] The C3 protein is in a biologically inactive state until the binding sites are exposed to the pathogenic cell surface and other complement components.[50] The complement system is frequently activated in many inflammatory diseases, including AMD.[48, 51] The complement system plays an important role as the first line of defense against the innate response, protecting the human organism by recognizing and mediating the removal of pathogens, debris, and dead cells; hence, therapies targeting complement C3 still need to be considered carefully in AMD patients.[48, 52]
It is worth noting that LCN1 also demonstrated importance in PPI. LCN1 is an oxidative stress-induced scavenger of potentially harmful lipid peroxidation products.[53] At present, oxidative stress is believed to be involved in the pathological process of AMD, although the specific mechanisms remain unclear,[54] and oxidative stress-related genes are also associated with AMD risk.[55, 56] Therefore, LCN1 is considered to have a protective role in the progression of AMD. In a recent study, LCN1 in tears demonstrated its potential as a biomarker in screening diabetic retinopathy,[57] which brings expectations for LCN1 to become a biomarker of AMD.
The primary limitation was the heterogeneity among enrolled studies. For example, different studies had different definitions of DEPs, which mainly reflected in the minimum fold change values of proteins to be displayed and the indicators for measuring the accuracy of results (p value or adjusted p value). Heterogeneity was also observed in the cohorts’ characteristics, such as the type of AMD and sociodemographic variables such as age or gender balance. Although this study included observational and randomized controlled trial (RCT) studies, it did not bring any additional heterogeneity because only statistics before intervention of RCT studies could be included in the meta-analysis, and the criteria for selecting patients were similar in the included RCT and observational studies.
In conclusions, various pathways associated with AMD have been elucidated, including lipid metabolism, the complement system, oxidative stress, inflammation, immunology, iron metabolism and ferroptosis. Four possible biomarkers, TF, APOA1, C3 and LCN1, were found to be significant in the pathogenesis of AMD and need to be further validated. These proteins should be further studied in larger cohorts to evaluate their potential for disease diagnosis and intervention.