In silico analysis of the molecular machinery underlying aqueous humor production: potential implications for glaucoma

Abstract

Background: The ciliary body epithelia (CBE) of the eye produce the aqueous humor (AH). The equilibrium between the AH production by the CBE and the outflow through the trabecular meshwork ultimately determines the intraocular pressure (IOP). An increased IOP is a major risk factor for primary open angle glaucoma (POAG). This study aims to elucidate the molecular machinery of the most important function of the CBE: the AH production and composition, and aims to find possible new molecular clues for POAG and AH production-lowering drugs.

Methods: We performed a gene expression analysis of the non-pigmented (NPE) and pigmented epithelia (PE) of the human CBE of post mortem eyes. We used 44 k Agilent microarrays against a common reference design. Functional annotations were performed with the Ingenuity knowledge database.

Results: We built a molecular model of AH production by combining previously published physiological data with our current genomic expression data. Next, we investigated molecular CBE transport features which might influence AH composition. These features included caveolin- and clathrin vesicle-mediated transport of large biomolecules, as well as a range of substrate specific transporters. The presence of these transporters implies that, for example, immunoglobins, thyroid hormone, prostaglandins, cholesterol and vitamins can be secreted by the CBE along with the AH. In silico, we predicted some of the molecular apical interactions between the NPE and PE, the side where the two folded epithelia face each other. Finally, we found high expression of seven POAG disease genes in the plasma membrane of extracellular space of the CBE, namely APOE, CAV1, COL8A2, EDNRA, FBN1, RFTN1 and TLR4 and we found possible new targets for AH lowering drugs in the AH.

Conclusions: The CBE expresses many transporters, which account for AH production and/or composition. Some of these entries have also been associated with POAG. We hypothesize that the CBE may play a more prominent role than currently thought in the pathogenesis of POAG, for example by changing the composition of AH.

Figure 1

Flow diagram. Schematic overview of the subdivisions made in the gene expression data of the NPE and PE into different sub-datasets.

Figure 2

Hypothetical molecular model of aqueous humor production. On the left, there is the physiological model of aqueous humor (AH) production of Civan and coworkers [2]. This model includes the different ion-channels involved in the AH production. On the right, there is hypothetical in silico molecular model of AH production. This model is based on our gene expression data of the ciliary body epithelia (CBE) and our strict selection criteria of the 10% highest and significant different expressed genes of the non-pigmented (NPE) and pigmented epithelia (PE) (all together in the Transport sub-dataset; see Methods). For the Na⁺/K⁺/2Cl^- symporter (coded by SLC12A1 and SLC12A2) we did not found a coding gene in the Transport sub-dataset (indicated with * in the model). Genes coding for ion-channels with moderate expression levels (for example SLC12A2) can be found in Table 1. The underlined genes (ATP1B1, GJA4, KCNB2, KCND2) are signature genes of the PE compared to the NPE (fold change >2.5 and p-value < 0.01; see Methods section).

Figure 3

Immunohistochemistry of caveolin 1 and clathrin in the ciliary body. Immunofluorescence staining for caveolin 1 (CAV1) and clathrin (CLTR) in human ciliary body sections. Our gene expression data show that CAV1 is significantly different expressed between NPE and PE (p-value < 0.01), with higher expression in NPE and all CLTR coding genes (CLTA, CLTB and CLTC) are highly (>90^th P) expressed in both NPE and PE. In this immunofluorescence picture, CAV1 is clearly present in the non-pigmented epithelium (NPE) and possibly also in the pigmented epithelium (PE). We found CLTR in both NPE and PE, especially in the apical plasma membrane. Both CAV1 and CLTR were found in the vascular endothelium of the stroma. Negative controls were for both proteins negative (not shown). Legend: blue = dapi = cell nucleus; red = cy3 = protein of interest.

Figure 4

Molecular interactions in the apical extracellular space of the pigmented (PE) and non-pigmented epithelium (NPE). Molecular network build in Ingenuity (http://www.Ingenuity.com), representing entries that are highly expressed and secreted in the apical extracellular space between NPE and PE. The top functions assigned to this network by Ingenuity were “Neurological disease, psychological disorders and cellular movement”. This network is contains many entries involved in matrix assembly and cytoskeleton organization (COL18A1, CTGF, DCN, FBN1, FBLN1, LTBP1, SPARC, and SPP1) and molecules involved in growth and proliferation (CTGF, EGF, IGF1, IGF2, IGFBP2-6, LTBP1, PTN, TGFB2 and VEGFA). IGF1 is a signature gene of the PE (yellow symbol). Together, these molecules are likely to be involved in the local turnover of the ECM between the PE-NPE layers of the CBE. We also identified several entries within the PE-NPE apical extracellular space of the CBE that are involved in amyloid-beta metabolism and plaque formation (A2M, APOE, CLU, SERPINA3, and TTR). Legend of different lines: Circles on top indicate direct relationships of entries with itself. Solid lines indicate that binding of the two proteins occur, arrows indicate that the first protein interfere with the expression or activity of the second protein.

Figure 5

Molecular interactions in apical transport of the ciliary body epithelia. Molecular network build in Ingenuity (http://www.Ingenuity.com), representing possible transporters in the apical plasma membranes of the pigmented and non-pigmented epithelia that transport the apical extracellular excreted entries outlined in Figure 4. The top functions assigned by Ingenuity to this network were “Neurological disease, psychological disorders and cell-to-cell signaling and interaction”. The major transport mechanisms to which these genes were connected were vesicle-mediated transporters (ANXA7, CAV1, CLTA, CLTC, and SORL1) and lipid (ABCA1, LDLR and SCARB1) and xenobiotic (ABCC5 and ABCG2) transporters. Two PE signature genes, ABCA1 and SLC40A1 (yellow symbols) are involved in this network. Legends of different lines: see Figure 4.