Avascular tissues of the anterior chamber receive their external cues from components of aqueous humor. Changes in protein or ionic concentrations within aqueous humor may have significant effects on cellular function and cell–matrix communication. However, only approximately 150 proteins have been identified in hAH. Using an approach that included multiple proteomic techniques and multigroup comparisons, we have identified 676 nonredundant proteins in hAH. More than 80% of the proteins are novel identifications for aqueous humor. To date, this study provides the most comprehensive list of proteins present in hAH.
Aqueous humor maintains a normal homeostatic environment and is essential to the proper functioning of anterior chamber tissues. Therefore, it is not surprising that most of the proteins identified by nano-LC-ESI-MS/MS have catalytic and enzymatic functions. For example, one of the functions of aqueous humor is to maintain a pathogen-free environment. Being immunoprivileged, the anterior chamber relies on complement to successfully rid the chamber of pathogens. Our proteomic data supports the presence of both the classic and alternative complement pathways. In all, 23 complement proteins were identified in hAH including C1q, C1r, C1s, C2–9, and complement regulatory molecules such as CD59; complement B, D, H, and I; and C1 inhibitor. The balance between complement activation molecules and complement regulatory molecules is important in maintaining healthy anterior segment tissues and avoiding autoimmune reactions that would significantly alter the function of these tissues.
Catalytic proteins such as type IV collagen are principal components of basement membranes36
and are one of the major extracellular matrix proteins upregulated during glaucoma. Other catalytic enzymes found in hAH, such as lactate dehydrogenase, have already been suggested to have a role in hAH outflow regulation, while showing a strong presence in the uveoscleral tissue.37
Several respiratory pathway catalytic enzymes such as aldolase and ketolase were also found in hAH and may have been secreted into the hAH by cells bathed by the fluid. The presence of strong angiogenic inducers as angiogenin and angiogenic inhibitors PEDF,38
type IV collagen,39
and vitamin D binding protein,40
suggests the presence of an equilibrium within hAH between proangiogenic and antiangiogenic molecules. The balance between angiogenic and antiangiogenic proteins may be important in the pathogenesis of anterior segment diseases.
Our antibody-based protein array study served the purpose of identifying proteins in the hAH that are present in low abundance and therefore are difficult to identify by nano-LC-ESI-MS/MS. Several members of the transforming growth factor β, tumor necrosis factor, fibroblast growth factor, interleukin, and growth differentiation factor families were identified. In addition, numerous growth factor and cytokine receptors were found in hAH, including receptors of the C-C chemokine, tumor necrosis factor, and interleukin superfamilies. In other biological fluids, circulating and soluble receptors play an important role in regulating growth factor and cytokine activity. Soluble receptors are normal components of body fluids in healthy individuals and levels of these receptors can modulate various growth factor and cytokine activities.41
For example, ligand binding to soluble receptors can protect the ligand from degradation, inhibit the ligand from initiating a signaling cascade or act in an agonistic manner. In the case of IL-6, the binding of IL-6 to its soluble receptor can stimulate cells that do not normally express an IL-6 receptor.42,43
Analysis of soluble receptors in hAH isolated from anterior chamber disorders, such as corneal dystrophies and glaucoma, may serve, not only as markers for the disease but as therapeutic targets in treating the disorder.
An important question in the midst of the description of the hAH proteome is “where are all the proteins found in hAH coming from?” Although hAH is considered to be a plasma filtrate, there are considerable differences between plasma and hAH that suggest several cells and tissues from the anterior segment may be involved in active secretion of ions/proteins into hAH.9–11
Bovine and primate studies suggest that hAH proteins originate in the ciliary body capillaries and move via a protein gradient toward the iris root where they diffuse through the ciliary epithelium into hAH.2,44,45
However, other studies have suggested that the ciliary epithelium and the pigmented and nonpigmented cells of the ciliary body are actively involved in pumping out regulatory proteins into the hAH possibly in conjunction with tissues surrounding the anterior/posterior chambers.46,47
These proteins include hemopexin, ceruloplasmin, ferroxidase, and glutathione S
-transferase, enzymatic proteins involved in detoxification and oxidative damage protection.48
Although many of the proteins identified may come from blood, cDNAs encoding plasma proteins have also been identified in the ciliary body.46
Among these are complement component C4, α2-macroglobulin, and the plasma form of glutathione peroxidase.46
Our study confirmed their presence in human hAH and suggests that the ciliary body may be one of the tissues in the anterior chamber that has the ability to produce and secrete traditional plasma proteins into hAH.
hAH samples obtained from patients undergoing cataract removal has been the traditional control in studies of hAH from patients with anterior chamber disorders, such as uveal melanoma, myopia, corneal rejection, and glaucoma.25–27,49,50
Although considered normal, the presence of a cataract affects the concentration and components of hAH as suggested by the increase in α-antitrypsin, α2-macroglobulin, and β-crystallin proteins.51,52
Although hAH collected from normal healthy adults would have been ideal, such samples cannot be obtained ethically.53
Therefore, it is possible that some of the proteins identified in our study are present because of the underlying cataract condition. Another limitation of this study was that our growth factor, cytokine, and receptor analysis was limited to the proteins that were present on the protein array. Therefore, not all growth factors, cytokines, and receptors in hAH were identified. Although our study was thorough, 676 proteins are probably only a fraction of the overall protein profile within hAH. Nevertheless, our study provides a comprehensive list of the hAH proteins. This list may be considered as a reference to look for differences in protein expression in various pathologic conditions of the anterior segment with the possibility of identifying novel biomarkers for the disease and possible targets for novel therapeutic treatments.