1. Introduction
Diabetic retinopathy (DR), the most common cause of vision loss in working-age adults in the US, presents a daunting challenge in both its pathology and available treatments [
1]. Despite extensive research, the precise mechanisms triggering its onset and progression remain elusive. Factors such as prolonged hyperglycemia, the generation of pro-inflammatory and pro-angiogenic proteins, lipids, and metabolites, and oxidative damage contribute to the impairment of retinal blood vessels [
2,
3,
4,
5]. The lack of a comprehensive understanding of its pathology hampers the development of effective therapies. Current treatments such as vascular endothelial growth factor (VEGF) inhibitors and corticosteroids primarily target late-stage manifestations to manage macular edema and neovascularization [
6]. However, these interventions often come after irreversible damage has occurred [
6,
7]. Novel diagnostic tools, including optical coherence tomography angiography (OCT-A), show promise in early detection, yet the challenge persists in preventing disease progression [
8]. Addressing the complex pathology of DR demands concerted efforts in unraveling its molecular underpinnings to pave the way for innovative therapeutic strategies aimed at halting its advancement from the early stages.
The vitreous humor, a vital gel-like substance bathing the posterior segment of the eye, is essential for maintaining ocular structure and function [
9]. Its composition reflects a delicate equilibrium of proteins, electrolytes, and growth factors, crucial for ocular health. Previous research has provided valuable insights into the dynamic nature of the vitreous composition, highlighting its role in intraocular pressure regulation, retinal support, and nutrient transport [
10,
11,
12,
13,
14]. However, in the context of diabetes mellitus, a metabolic disorder characterized by hyperglycemia, the vitreous undergoes significant alterations, reflecting the pathogenesis of DR, a leading cause of vision loss globally [
15,
16,
17]. The multifactorial nature of DR involves complex interplays of biochemical pathways, with vitreous composition playing a pivotal role [
18,
19].
Recent studies have elucidated the presence of inflammatory cytokines, angiogenic factors, and extracellular matrix proteins in the vitreous of diabetic individuals, underscoring their involvement in retinal damage and neovascularization [
17,
19]. Advances in proteomic techniques have facilitated the identification of novel biomarkers associated with DR progression [
19,
20]. Despite these advancements, the available data often combine results from both type 1 and type 2 diabetic patients, along with compounding factors such as diverse treatments and co-morbidities. A comprehensive understanding of vitreous composition in type 2 diabetes remains unclear, necessitating further investigation.
Comparative studies analyzing vitreous humor from diabetic and non-diabetic individuals are crucial for deciphering the molecular mechanisms underlying DR and identifying potential therapeutic targets. Therefore, in the present study, we aimed to address this gap by conducting a detailed liquid chromatography–mass spectrometry (LC–MS) proteomic analysis of vitreous humor samples sourced from both normal subjects and patients with type 2 diabetes mellitus. Through rigorous examination of protein profiles and inflammatory mediators, we endeavored to delineate the distinct biochemical signatures associated with diabetes and their implications for DR pathogenesis.
4. Discussion
DR is a leading cause of vision impairment and blindness in diabetic patients worldwide, driven by chronic hyperglycemia, inflammation, and associated metabolic disturbances [
1,
2]. Understanding its molecular mechanisms is crucial for developing effective therapies. Proteomic analysis of vitreous humor has emerged as a powerful tool for identifying biomarkers and elucidating the pathophysiology of DR [
18,
19]. Recent advancements in mass spectrometry and bioinformatics enable comprehensive profiling of the vitreous proteome, providing insights into inflammation, angiogenesis, and extracellular matrix remodeling in proliferative DR [
25,
26]. However, apart from smaller sample sizes and technical variability, other limitations of these studies include compounding factors from different cohorts of patient characteristics and treatments of various conditions they received. Our study focuses on proteomic analysis of vitreous samples from diabetic and non-diabetic subjects, revealing proteins significantly associated with metabolic regulation and inflammation, and highlighting alterations in pathways such as Wnt and cytokine-induced inflammation. This multifaceted approach enhances our understanding of possible pathways involved in DR development and aids in identifying novel therapeutic targets.
The comprehensive gene enrichment analysis of differentially expressed proteins in the diabetic vitreous humor versus non-diabetic vitreous revealed important insights into how diabetes affects vitreous content. Principal component analysis (PCA) demonstrated a clear separation between diabetic and non-diabetic groups based on protein expression variances, underscoring the distinct proteomic alterations associated with DR and aligning with previous studies that have reported significant changes in the vitreous proteome of DR patients [
27,
28,
29]. We identified 22 differentially expressed proteins, with 12 upregulated and 10 downregulated in diabetic samples. Notably, proteins such as nuclear envelope integral membrane protein 2 (NEMP2), zinc finger protein 814 (ZNF814), oligodendrocyte-myelin glycoprotein (OMG), coronin-1A (CORO1A), inositol 1,4,5-trisphosphate receptor type 2 (ITPR2), protocadherin-23 (DCHS2), calcium homeostasis endoplasmic reticulum protein (CHERP), and myosin-2 (MYH2) were significantly increased in diabetic vitreous samples. Although the direct involvement of any of these proteins in retinal injury and inflammation has not yet been investigated, pathways involving some of these proteins are implicated in various cellular processes, including membrane integrity, signal transduction, myelination, and calcium homeostasis, which are crucial in the pathophysiology of DR [
29,
30,
31,
32,
33,
34]. These findings are also consistent with existing literature that highlights inflammation, immune response, and metabolic dysregulation as key factors in DR progression. For instance, the upregulation of ITPR2 and CHERP suggests altered calcium signaling and homeostasis, which have been implicated in retinal cell death and vascular dysfunction in DR [
12]. Similarly, increased levels of OMG and CORO1A align with previous reports of neuroinflammatory responses and cytoskeletal reorganization in DR [
29]. The identification of these differentially expressed proteins may provide as valuable biomarkers for DR and potential targets for therapeutic interventions. Future studies focusing on validating these findings in larger, more diverse cohorts and exploring the functional roles of these proteins in DR pathogenesis is warranted.
Conversely, proteins such as Immunoglobulin heavy variable 3-64D, mRNA cap guanine-N7 methyltransferase, Adhesion G protein-coupled receptor B3, Kinesin-like protein KIF1B, Aldehyde dehydrogenase (mitochondrial), Dipeptidyl peptidase 4, Triokinase/FMN cyclase, G-protein-signaling modulator 1, Protein argonaute-1, and Plexin-B3 were expressed at lower levels in diabetic vitreous. Some of these downregulated proteins are associated with pathways that regulate the immune response, mRNA processing, cell adhesion, intracellular transport, mitochondrial dysregulation, and metabolic regulation [
25,
35,
36], indicating potential disruptions in these pathways in DR.
Our pathway enrichment analysis of significantly altered proteins in the vitreous humor of diabetic individuals has revealed key insights into the molecular mechanisms underlying DR. Using databases such as Panther, KEGG, Reactome, and BioCarta, we identified 26 dysregulated pathways within the diabetic vitreous. The top KEGG pathways implicated include glycerolipid metabolism, pantothenate and CoA biosynthesis, histidine metabolism, and ascorbate and aldarate metabolism, highlighting the profound metabolic alterations in DR. These findings align with previous studies that have reported metabolic dysregulation as a hallmark of DR pathogenesis [
19,
29].
In the Panther database, several key signaling pathways were identified, such as Wnt signaling, heterotrimeric G-protein (Gαq and Gαo)-mediated pathways, chemokine and cytokine-mediated inflammation, and histamine H1 receptor-mediated signaling. These pathways are crucial for cellular communication and inflammatory responses, which are known to be also disrupted in DR [
30]. The involvement of inflammatory signaling pathways underscores the role of chronic inflammation in retinal damage and progression of DR, consistent with previous literature that highlights inflammation as a central component in DR [
12]. BioCarta pathway analysis, although revealing fewer altered pathways, identified critical processes such as the cycling of Ran in nucleocytoplasmic transport [
37,
38,
39] and the role of PI3K subunit p85 in regulating actin organization and cell migration [
40]. Therefore, these pathways are pertinent to retinal pathogenesis as they are involved in maintaining cellular structure and facilitating cellular responses to stress. The disruption of these pathways could lead to cytoskeletal abnormalities and impaired cellular migration, contributing to the retinal alterations observed in DR. Reactome enrichment analysis further supported the involvement of several regulatory pathways, including transcriptional regulation by MECP2, Ca
2+ signaling, regulation by small RNAs, fructose catabolism, and MET/PTPN11 and MET/TNS protein signaling. The dysregulation of these pathways suggests a complex interplay between genetic regulation, calcium homeostasis, and metabolic pathways in the progression of DR [
41,
42]. The significant alteration in transcriptional regulation and calcium signaling aligns with the findings of previous studies that have linked these processes to neurodegeneration and vascular dysfunction, including retinal [
43,
44,
45,
46]. Overall, these results provide a comprehensive overview of the dysregulated pathways in diabetic vitreous humor, reinforcing the multifactorial nature of DR. These findings, however, highlight the need for further research to validate these pathways and explore their potential as therapeutic targets.
Our study comes with several limitations. First, the sample size is relatively small, preventing a full analysis of the patient characteristics, including the identification of the differences in risk factors associated with patients. Thus, a larger sample size is needed to detect such significant differences. Second, the vitreous samples were collected postmortem, and some of the patients had other diseases, such as heart disorders, which could have influenced the gene ontology analysis, such as molecular functions and biological processes, leading to potentially irrelevant findings regarding eye diseases. Third, though some of the patients had around 20 years of being diabetic, the development of DR is not confirmed (or data are unavailable) but is expected to occur based on the duration of diabetes. Fourth, the samples were taken from only type 2 diabetic patients; thus, type 1 diabetes was not feasible in our analysis. Finally, the exact role of each differentially expressed protein that is either highly or lower expressed in the context of diabetes deserves further experimental consideration, depending on the significant change levels and contributions to the pathogenesis of eye diseases.