Abstract
Purpose :
Several studies have pointed out phenotypical alterations of circulating immune cells in subjects diagnosed with glaucoma and increased plasma levels of pro-inflammatory mediators, but the molecular alterations have never been explored. We have here undertaken a shot-gun proteomic study of Peripheral Blood Mononuclear Cells (PBMC) isolated from subjects diagnosed with primary open angle glaucoma (POAG) and cataract (controls) to figure out molecular pathways of pathological relevance.
Methods :
The study was authorized by the local ethic committee. PBMCs (n=17 POAG, n=17 cataract; age: 69.2±5.0y POAG, 70.3±9.8y, cataract; comparable gender ratio) were isolated from peripheral blood by Ficoll-histopaque sedimentation and lysed in urea buffer. 200 µg of proteins were reduced, alkylated and digested with trypsin (1:50 ratio). Peptides (750 ng) were injected in a Orbitrap Exploris 240 online with a nanoUHPLC and analysed in DDA and DIA mode (label free quantification). Proteins were searched by Proteome Discoverer and FragPipe (DIA-NN library-free) and output data analysed by R.
Results :
Data commented below refers to the DIA output (DDA data were overlapping). Quality controls (e.g., Coefficient of Variation, blood proteins contamination, etc.) were checked as optimal.
Proteins were filtered for identification with at least 2 peptides and in at least 70% of POAG and cataract PBMCs.
4322 proteins identified and quantified were common to POAG and cataract; 414 proteins were exclusive of POAG and 143 of cataract.
Volcano plot (log2_Fold Change>0.5, p<0.05) indicated that 259 proteins were upregulated in POAG and 117 in cataract.
GeneOntology-enrichment showed that POAG PBMCs suffered from possible alterations (the list is not exhaustive) of: humoral immune responses, mitochondria homeostasis (e.g., redox enzymes), proteases (e.g., MMPs), macroautophagy (e.g., Beclin1), inflammatory cascades (e.g., NF-kB) and apoptosis (e.g., Bcl-2). Main targets were studied by Western blotting to confirm proteomics data.
Conclusions :
Proteomic datasets suggest that glaucoma PBMCs are charged with dysregulation of key intracellular pathways. Further studies are needed to figure out the distribution of cell subsets, their polarization and metabolic trajectories along with the role of these factors in mediating the neuroinflammation state that accompanies glaucoma progression.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.