MS-based proteomics is a popular method for interrogating the composition of vitreous in retinal disease states, including PDR. Prior shotgun proteomic studies of PDR vitreous vary greatly in the sample sizes used, which range from one to 74 samples per group (16, 43); MS methods chosen; and number of proteins identified, ranging from as few as 11 to over 2,400 (16, 44). This study aimed to develop and validate a feasible, rigorous, and scalable method for vitreous proteomic studies through assessment of variability and determination of power. Pathway analyses to infer biological meaning revealed previously unknown alterations that may be implicated in PDR pathogenesis.
Technical variability was nearly absent when performing TMT-MS using a single 10-plex and remained minimal when using multiple plexes. Biological variability was greater than technical variability, as expected, but remained quite low. Normalizing across plexes did not reveal any evident plex bias, underlining the feasibility of applying the described normalization methods to studies examining samples distributed across multiple plexes. Given the number of samples per group required to achieve acceptable power, TMT multiplexing will be critical to scale up experiments while minimizing batch effects. Bridging across plexes using pool samples may prove to be an integral technique realize sufficient power for differential expression analysis in inherently noisy proteomic data with lower biological effect sizes. Samples within a disease phenotype or subphenotype were similar to one another. One concern that has arisen frequently in vitreous proteomic studies utilizing PDR samples is that blood contamination may skew results (45–48). A prior study addressed this concern by excluding samples with hemoglobin concentrations > 5 mg/mL (equal to 0.5 g/dL) (47). Given this concern, we grouped PDR subphenotypes according to hemoglobin concentration in non-depleted vitreous. Samples that were visibly tinted yellow or red had hemoglobin concentrations no higher than 0.0084 g/dL. Therefore, our hemoglobin cutoff is more conservative than what has been reported in prior literature. To further address the concern of blood contamination, we visualized the distribution and hierarchical clustering of abundant plasma proteins and proteins highly expressed in erythrocytes relative to other cell types. No discernable pattern was seen in the distribution of the proteins across samples, phenotypes, or subphenotypes, and these proteins did not appear to drive sample clustering in any way. Thus, it is unlikely that blood contamination contributed in any significant way to the vitreous proteome in PDR.
Due to the high degree of similarity in terms of pathways differentially expressed across the three PDR subphenotypes compared to controls and the above findings on power, the following discussion focuses on the overall comparison group of all PDR samples versus all controls. Only those pathways with statistically significant differential expression and/or activation status were included in the analysis, defined as p-value < 0.05 and z-score ≥ magnitude 2. Differential expression is reported according to the overrepresentation p-value generated by iPathway guide, while activation status is given in terms of z-score generated by IPA.
Pathways centering on metabolism were both overrepresented and activated in PDR vitreous relative to controls. “Metabolic pathways”, “carbon metabolism”, and “glycolysis / gluconeogenesis” were differentially expressed in PDR vitreous relative to controls, with the vast majority of pathway components being upregulated (Table 3). In addition to being overrepresented, glycolytic and gluconeogenic pathways were predicted to be activated (Table 4).
Carbohydrate metabolism, including glycolytic and gluconeogenic pathways, is dysregulated in diabetes. Glycolysis converts glucose into lactate and releases ATP and reducing equivalents, whereas gluconeogenesis is a reversal of this pathway that generates glucose from non-carbohydrate precursors. These pathways are encompassed within “carbon metabolism”, which also includes other carbon utilization pathways such as the pentose phosphate pathway and citric acid cycle. “Metabolic pathways” encompasses all of the differentially expressed genes in “glycolysis/gluconeogenesis” and “carbon metabolism.”
The duration and degree of hyperglycemia in persons with diabetes are associated with the development and progression of DR (49), and intensive management of blood glucose reduces the risk of DR development and progression (50–58). Recent evidence indicates that abnormal flux through the glycolysis pathway leads to the activation of several pathways known to be involved in the pathogenesis of complications of diabetes. Direct assessment of retinal metabolism using radiolabeled glucose revealed modest upregulation of glycolysis in the BKS db/db mouse model of type 2 diabetes at 24 weeks of age (59). However, similar studies in a rat model of insulin-deficient diabetes showed no meaningful increase in retinal glycolysis (60). The current study is the first to analyze human ocular metabolic responses to long-term diabetes. The overrepresentation of metabolic pathway members in PDR vitreous may similarly indicate disruption of glucose homeostasis due to hyperglycemia.
A prior proteomic study of PDR vitreous by Gao et al. identified elevated the “metabolic pathway” components carbonic anhydrase 1 and 2 (CA1, CA2) (46), which reversibly hydrate carbon dioxide as part of pH and fluid balance and were the most upregulated proteins in this pathway in the current study. Gao et al. found levels of CA1 and CA2 to be several times higher in PDR vitreous relative to that of non-diabetic controls. Further analyses of CA1 and CA2 indicated they may increase permeability of the retinal vasculature, with the actions of CA1 being additive to those of vascular endothelial growth factor (VEGF) (46). Thus, CA1 and CA2 may represent specific metabolic pathway members that contribute to PDR pathogenesis.
“Protein kinase A (PKA) signaling” was also activated in PDR vitreous. This pathway has diverse regulatory activity, modulating growth and development, memory, and metabolic functions. PKA regulates angiogenesis in the developing retina; its inhibition in mice caused vascular defects via an increase in the number of endothelial tip cells, resulting in hypersprouting (61). Similarly, a later study demonstrated that PKA reduced endothelial sprouting capacity (62). These angiogenic regulatory effects may reflect an attempt by the retina to modulate revascularization in the setting of DR. PKA-dependent pathways also augment retinal ganglion cell regeneration (63).
“Nuclear factor-erythroid 2-related factor 2 (Nrf2)-mediated oxidative stress response” was activated in PDR vitreous relative to controls and is a widely studied mediator of the cellular response to oxidative stress. Nrf2 is a transcription factor that, when activated, leads to transcription of antioxidant enzymes and other proteins involved in detoxification. Because of the high rate of oxygen consumption in the retina relative to other tissues, it is especially vulnerable to oxidative stress (64, 65). Given this vulnerability, it is not surprising that oxidative stress is involved in various mechanisms underlying DR pathogenesis (66, 67). Reactive oxygen species (ROS) are the main source of oxidative stress and are produced physiologically during carbohydrate metabolism. When the production of ROS cannot be balanced with antioxidant mechanisms, ROS accumulate and induce DNA damage and inflammation, stimulating VEGF production (67). Nrf2 signaling serves as an attempt to offset these pathological changes. Xu et al. demonstrated that knockout of Nrf2 in a diabetic mouse model resulted in early blood-retina barrier dysfunction and declining neural function (68). A study of diabetic rats and human donor retinas demonstrated increased Nrf2 levels, but decreased Nrf2 activity due to increased binding with its inhibitor, Keap1, preventing its translocation to the nucleus for transcription of antioxidant response elements (69). The increased activation of Nrf2 signaling in PDR vitreous may indicate an effort, though possibly unsuccessful, to restore the balance of antioxidant molecules in the context of increased ROS.
“Serine protease inhibitor, Kasal type 1 (SPINK1) pancreatic cancer pathway” was activated in PDR vitreous. SPINK1 is known for its role in inhibiting pancreatic trypsin in cases of premature trypsinogen activation and its role in familial forms of pancreatitis. More recently, however, SPINK1 has been recognized as a possible acute phase reactant (70) and growth factor, and has specifically been shown to stimulate endothelial cell growth (71). Though the role of SPINK1 signaling in the retina is yet unknown, a recent study demonstrated a higher incidence of DR in individuals with fibrocalculous pancreatic diabetes, a form of diabetes mellitus often associated with SPINK1 mutations, relative to individuals with type 2 diabetes mellitus (72). Further research is needed to elucidate the role of SPINK1 in DR.
In contrast to activated pathways, “semaphorin neuronal repulsive signaling pathway” was inactivated in the PDR group relative to controls. Specifically, the semaphorins sema3A, sema3F, and sema6A were present at decreased levels. Semaphorins have dual roles in regulating both repulsive neuronal guidance during development and angiogenesis. Sema3A and sema3F serve an anti-angiogenic role in retinal and other tissues. Sema3A is produced by retinal ganglion cells under hypoxic conditions and decreases endothelial cell migration, directing neovascularization away from ischemic retina and toward the vitreous. However, intravitreally delivered recombinant sema3A prevents neovascularization into the vitreous (73). Thus, the location of sema3A determines the direction of its anti-angiogenic effects. The decreased levels of sema3A in PDR vitreous in the current study are in line with these findings, as PDR is characterized by neovascularization into the vitreous. Sema3F has also been shown to have anti-angiogenic functions, mainly in the outer retina, where it is almost singularly expressed. Reduced sema3F levels have been identified in retinal pigment epithelium derived from human donors with a history of neovascularization of the outer retina (74). Additional studies are needed to determine whether sema3F plays a role in neovascularization of the inner retina, as occurs in PDR. Recently, sema3F was shown to suppress VEGF-induced endothelial cell proliferation with a higher efficacy than anti-VEGF antibody treatment; this effect was observed at a sema3F concentration 10-fold lower than that of VEGF (75). Sema6A also decreases endothelial cell migration in a dose-dependent manner (76). Decreased semaphorin signaling in PDR vitreous may be one factor permitting the extension of retinal neovascularization into the vitreous.
“Interleukin (IL)-15 signaling” activation was decreased in PDR vitreous relative to controls. IL-15 regulates natural killer cells and T lymphocytes and is produced by diverse cell types, including macrophages, fibroblasts, and nerve cells. More recently, roles for IL-15 in metabolism have been elucidated, specifically in the context of obesity. Obesity is a key player in the development of insulin resistance, a phenomenon that characterizes the pathogenesis of type 2 diabetes mellitus. Sun and Liu found that transfer of the IL-15 gene in high-fat diet-induced obese mice prevented weight gain, lessened the development of hepatic steatosis, and improved glucose homeostasis (77). Transfer of the IL-15 gene along with its soluble receptor had the same effects, along with improving insulin sensitivity (78). Similarly, Barra et al. (79) found that delivery of the IL-15 gene to high fat diet-induced obese mice increased sensitivity to insulin and better responses to a glucose challenge relative to both untreated high-fat diet mice and low-fat diet lean controls. IL-15 may also have insulin-independent effects on glucose metabolism. A recent study demonstrated that IL-15 improved glucose metabolism by activating AMP-activate protein kinase (AMPK) and increasing glucose transporter type 4 (GLUT4) translocation to the skeletal muscle membrane (80). AMPK-mediated GLUT4 translocation is induced by exercise in a mechanism independent of insulin (81, 82) and thus may mitigate the effects of insulin resistance. Further research is needed in order to determine whether these properties are generalizable to retinal tissue, but it is possible that the decrease in IL-15 signaling reflects the impaired glucose and insulin responses that ultimately precipitate diabetes complications such as retinopathy.
“Liver X receptor (LXR)/retinoid X receptor (RXR) activation” was also inhibited in PDR vitreous. RXRs and LXRs are nuclear receptors that form heterodimers to exert transcriptional regulation. RXRs facilitate the actions of retinoids, while LXR acts to increase cholesterol efflux. The LXR/RXR heterodimer has regulatory functions on both metabolic and inflammatory processes. Studies evaluating the actions of the LXR/RXR heterodimer in diabetes are lacking, but ample research exists on each individual receptor. Multiple studies have demonstrated a glucose-lowering effect of RXR agonists. RXR agonists have been shown to lower serum glucose levels (83, 84) and increase insulin sensitivity (84) in diabetic animal models. RXR also increases both insulin-dependent and independent glucose uptake in skeletal muscle (85). Given these glucose-lowering effects of RXR agonists, the decreased activity of RXR in PDR vitreous may contribute to dysregulated retinal glucose metabolism. LXR, a cholesterol-modulating receptor that also functions in inflammation, may influence the development of diabetes and its complications through actions on multiple cell types. In a mouse model of diabetes, an LXR agonist improved the function of endothelial cell precursors responsible for vascular repair and reduced expression of a marker of neurodegeneration (86), suggesting involvement in both vascular and neural effects of diabetes. LXR activation has also been shown to block hyperglycemia-induced endothelial cell senescence, potentially protecting against the atherosclerotic processes that are accelerated in diabetes (87). Treatment of diabetic animals with an LXR agonist resulted in neuroprotective effects (88), further suggesting a role in the neurodegenerative aspect of diabetes. In line with decreased activity of LXR/RXR activation in PDR vitreous in the current study, decreased LXR was observed in retinal tissue from both diabetic mice and diabetic human donors (89). The decreased LXR/RXR activation in PDR the current study is thus in line with the microvascular and neurodegenerative processes that characterize diabetic retinopathy.
“Synaptogenesis signaling pathway” was also inhibited in PDR vitreous. Synaptogenesis refers to the formation of neural synapses and is mediated by interactions between diverse adhesion molecules. In ocular development, synaptogenesis plays a role in synchronizing the timing of retinal synapse formation with eye opening (90). Studies specifically examining synaptogenesis in the adult retina in diabetes are lacking, but synaptogenesis is known to be a continuous and dynamic modulator of neural circuitry in the adult brain (91). Dysfunctional synaptogenesis is both a cause and an outcome of various neurodegenerative and neurodevelopmental central nervous system disorders (92). Neurodegenerative changes in the retina precede the microvascular injury of clinically detectable diabetic retinopathy (93–96). Consistent with prior literature, it is possible that decreased synaptogenesis signaling in PDR vitreous contributes to or results from the neurodegenerative changes that occur in early DR pathogenesis.
Collectively, these data reveal profound alternations in ocular metabolism, inflammatory processes, and neurotrophic pathways in the patients with PDR. These findings are consistent with the late stage of DR in which most of the patients had previously undergone panretinal laser photocoagulation and/or intravitreal anti-vascular endothelial cell growth factor treatments. These patients exhibit both vascular and neural retinal degeneration and have developed neovascular and fibrotic responses that lead to the need for therapeutic vitrectomy.
We carefully examined the potential role of blood on the proteome profile by phenotyping the samples by color and hemoglobin concentration. Remarkably, plasma and erythrocyte-derived did not distinguish PDR proteomic profiles from the non-diabetic controls. The process of depleting the most abundant plasma-derived proteins prior to MS analysis was a standard feature of our protocol and is important to enable detection of low abundance retina-derived proteins that are likely involved in the pathogenesis of PDR.
Potential role of extracellular vesicles
In addition to the components of the vitreous proteome discussed above, we have previously shown that human vitreous contains an abundant population of EVs (14). In the current study, EVs were more abundant in PDR vitreous than that of control, and distinct size populations varied with subphenotype. The increased population of larger diameter EVs seen across subphenotypes with increasing hemoglobin concentration may represent a uniquely functioning subset of EVs related to PDR pathogenesis or severity. EVs are secreted by all cell types and function in cell-cell communication, playing critical roles in both physiological and pathological processes. These vesicles contain diverse biomolecules indicative of the processes occurring in their parent cells at the time of secretion and are therefore rich with information about the tissues from which they derive (97, 98). Although the specific cells of origin of vitreous EVs remain unknown, it is plausible that they derive from multiple cell types in the adjacent tissues, including cells of the retina. Therefore, vitreous EV analysis may yield critical information about retinal disease states. Many of the above pathways have been shown to be mediated, in part, by EVs. For example, EVs play an emerging role in metabolism and metabolic disease. The majority of enzymes of the glycolysis pathway are among the top 100 most commonly identified proteins in exosomes (99), a subset of EVs. In certain systems, EVs independently generate ATP via glycolysis (100). EVs also impede insulin signaling and precipitate insulin resistance in adipose tissue (101, 102). Due to their high cholesterol content, EVs may serve as an additional strategy to reduce the cellular lipid burden in cholesterol-overloaded conditions, helping to preserve cholesterol homeostasis (103). Recently, EVs secreted by liver cells were recently shown to regulate adipose and lipid production in recipient adipocytes (104). In addition to metabolic functions, EVs play roles in angiogenesis via multiple pathways. EVs are able to both deliver angiogenic molecules, including VEGF, from cell to cell (105) and to induce expression of VEGF transcripts (106). An EV-associated form of VEGF was shown to possess increased potency in terms of VEGF receptor activation in recipient cells (107). Other angiogenic signaling molecules, such as the angiopoietins and Wnt pathway members, are also present in EVs (108–115). EVs also possess protective properties against oxidative stress. EVs have been shown to alleviate oxidative stress in animal models via Nrf2 activation (116) and by lessening myeloperoxidase and ROS activities (117). In addition to modulating oxidative stress, EVs also have the capacity to enhance or decrease inflammatory processes. EVs released by cells exposed to inflammation exert anti-inflammatory effects via the cyclooxygenase/prostaglandin E2 pathway (118). Contrarily, cells exposed to lipids released EVs that led to pro-inflammatory changes in recipient cells (119). Emerging evidence also points to a role for EVs in synaptic plasticity. Wnt pathway members are involved in synapse formation and plasticity, and Wnt members and their binding proteins are transferred across neuromuscular synapses in a mechanism requiring EVs (113). Other proteins with known roles in synaptic plasticity also require EVs for their trans-synaptic transport and function (120, 121). Thus, EVs are abundant in vitreous and play known roles in the processes reflected in the above pathway analysis, namely metabolism, angiogenesis, oxidative stress, inflammation, and synaptogenesis. Further investigation is needed to determine the functional role of EVs in the vitreous and the degree to which they may be involved in the pathways discussed here.
The current study underlines the importance of taking statistical power into account when designing vitreous proteomics studies. The use of mass spectrometry-based analysis enabled unbiased identification of signaling pathways, an advantage over prior studies of vitreous utilizing only targeted approaches such as enzyme-linked immunosorbent assays or western blots. This approach facilitated identification of both upregulated and downregulated pathways, the latter of which is often brushed over in the literature. The current study also has the advantage of including an activation z-score to show directional pathway involvement, whereas prior studies have focused mainly on pathway enrichment only. Further, the results presented here are the first to validate the use of bridging samples to scale up sample size to achieve sufficient power. These results also show that arranging such samples in plexes of 10 accomplishes comparable proteomic depth of coverage at 1/10th the cost of non-plexed experiments, increasing the feasibility of larger scale vitreous proteomics studies.
A limitation of this study is its examination of only a single retinal disease. Although the findings from these data can be applied to other diseases, precise determination of the ideal sample size for analysis of particular disease phenotypes will need to be assessed on an individualized basis. Nonetheless, the current data can serve as a useful guide for both interpreting the statistical rigor of prior vitreous proteomic studies and for estimating the necessary sample size in future studies.
Future studies are needed to determine whether sufficient power can be achieved at similar sample sizes in vitreous proteomic studies examining different retinal diseases.