Anti-VEGF therapy is commonly used to treat proliferative diabetic retinopathy (PDR), but the exact mechanism of VEGF signaling is not fully understood. Using data-independent acquisition mass spectrometry (DIA-MS), we analyzed proteomic changes in aqueous humor (AH) samples collected before and one week after intravitreal ranibizumab (IVR) treatment from 10 PDR patients to discover potential biomarkers. Resultantly, 875 proteins were quantified and 26 proteins were significantly altered in response to IVR treatment in PDR. Further investigation through gene ontology (GO) and pathway analysis revealed that these differentially expressed proteins were primarily involved in extracellular matrix (ECM) and platelet degranulation signaling. Protein-protein interaction analysis highlighted five hub proteins (COL3A1, DPT, VEGFA, SPP1, SERPING1) that were found to be ECM components. Enzyme-linked immunosorbent assay (ELISA) confirmed the decreased levels of VEGFA and increased levels of DPT proteins after IVR treatment in another 8 samples of AH in 4 PDR patients. Our study provided novel insights into aqueous proteins of PDR following IVR treatment. Targeting the ECM pathway, particularly the elevation of DPT protein, may provide a deeper understanding of the anti-VEGF resistance and VEGF signaling in PDR.

Abstract Purpose The main objective of this study is to characterize and analyze modified peptides in DBS samples. This includes deciphering their specific PTMs and understanding their potential impact on the population or disease cohort under study. Experimental design Using mass spectrometry-based proteomic approaches, we performed a comprehensive analysis of DBS samples. Our focus was on the identification and quantification of modified peptides. We also took advantage of recent advances in DBS mass spectrometry to ensure accurate detection and quantification. Results A comprehensive analysis identified 976 modified peptides in DBS samples. Of these, a subset of 211 peptides was consistently present in all samples, highlighting their potential biological importance and relevance. This indicates a diverse spectrum of PTMs in the proteome of DBS samples. Conclusions and clinical relevance Integration of mass spectrometry and proteomics has revealed a broad spectrum of modified peptides in DBS samples and highlighted their importance in biological processes and disease progression. Accurate detection of these PTMs may be critical for risk stratification and disease management. This study improves the understanding of molecular mechanisms underlying biological processes and disease development, providing important insights for clinical applications.

Acute glaucoma’s main sign is acute ocular hypertension (AOH), leading to retinal ganglion cell (RGC) death and irreversible visual loss. However, there is currently no approved effective therapy for this condition. This research aimed to identify the major regulators and the overall protein changes involved in AOH-induced RGC death. Mass-spectrometry was used to analyze proteomic patterns in the retinal protein extracts from the AOH and sham-group, and then Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway studies were performed. In proteomics analysis, we identified 92 proteins in the AOH group compared to the control group, with 58 proteins being up-regulated and 34 proteins being down-regulated. Western blot and biochemical assay analyses identified changes in Fatty acid-binding protein 7 (FABP7), and caveolin-1(Cav-1) that were related to fatty acid metabolism and ocular inflammatory signaling. Moreover, variations in the expression of the proteins Galectin-1 (Gal-1), S100 calcium-binding protein A6 (S100a6), and Visinin-like protein-1 (VILIP) was shown, all of which were associated to the process of neuronal ischemia. Our investigation demonstrated that neuroinflammation and fatty acid metabolism were involved in retinal impairment following AOH, offering a potential therapeutic strategy for acute glaucoma.

Venous Thromboembolism (VTE) remains a significant cause of morbidity and mortality worldwide. Rivaroxaban, a direct oral factor Xa inhibitor, mediates anti-inflammatory and cardiovascular-protective effects besides its well-established anticoagulant properties; yet, these remain poorly characterized. Extracellular vesicles (EVs) are considered proinflammatory messengers regulating a myriad of (patho)physiological processes and may be highly relevant to the pathophysiology of VTE. The effects of Rivaroxaban on circulating EVs in VTE patients remain unknown. We have established that differential EV biosignatures are found in patients with non-valvular atrial fibrillation anticoagulated with Rivaroxaban versus warfarin. Here, we investigated whether differential proteomic profiles of circulating EVs could also be found in patients with VTE. We performed comparative label-free quantitative proteomic profiling of enriched plasma EVs from VTE patients anticoagulated with either Rivaroxaban or warfarin using a tandem mass spectrometry approach. Of the 181 quantified proteins, 6 were found to be either exclusive to, or enriched in, Rivaroxaban-treated patients. Intriguingly, these proteins form a cluster tightly involved in negative feedback regulation of inflammatory and coagulation pathways, suggesting that EV proteomic signatures may reflect both Rivaroxaban’s anti-coagulatory and anti-inflammatory potential. These findings may be of translational relevance towards characterizing the emerging anti-inflammatory and cardioprotective mechanisms associated with this therapy.