Glucocorticosteroid-induced IOP elevation is a well-described side effect of steroid therapy in some individuals. Corticosteroids seem to affect IOP in a dose- and duration-dependent manner and can exert such an effect when administered by a variety of routes.
13 If the duration of corticosteroid therapy is lengthy it can lead to glaucomatous optic neuropathy.
4 The prevalence of steroid-induced glaucoma has been increasing over the past few years because potent, long-lasting steroids are increasingly used to treat many posterior pole conditions.
14,15 It has been suggested that a specific common relationship exists between this ocular response to corticosteroids and the factors in the TM that cause primary open-angle glaucoma.
16 Steroid-induced glaucoma is currently treated medically by either decreasing aqueous inflow (β-blockers, carbonic-anhydrase inhibitors, α2-agonists) or increasing uveoscleral (nontrabecular) outflow (prostaglandin analogs and α2-agonists). However, neither of these approaches addresses the underlying pathology at the level of the TM, because the underlying mechanisms for development of steroid-induced reduction in outflow facility remain largely unknown. Therefore, understanding the cellular and molecular processes that lead to corticosteroid-induced ocular hypertension is important because it may illuminate the mechanisms for primary open-angle glaucoma and lead to new therapies to lower IOP. The present work is a first attempt to understand the early molecular events that lead to steroid-induced IOP elevation.
Many investigators have studied the effects of glucocorticoids on the TM (reviewed in Wordinger and Clark
17 and Jones 3rd and Rhee
18 ). The work has used cultured TM cells,
19 –23 organ cultured eyes,
24 –30 and some in vivo models.
31 –33 These investigations have targeted morphologic changes, as well as gene expression, extracellular matrix (ECM), cytoskeleton, and cell adhesion molecules in the TM (extensively reviewed in Wordinger and Clark
17 and Borras
34,35 ). However, most of these studies are performed on cell or organ cultures that are not necessarily representative of the in vivo condition. For example, myocilin—one of the molecules associated with the development of glaucoma when mutated—is expressed in high amounts in trabecular cells in vivo,
36 but is barely expressed in vitro unless cultures are exposed to corticosteroids.
37 The few in vivo studies performed thus far have initially focused on anatomic alterations
38 and on candidate genes and their protein products.
39 A few global gene expression investigations have compared gene expression in organ-cultured TM between normal and glaucomatous eyes.
40,41 The present work represents the first time that global gene expression in the TM has been studied, shortly after the induction of elevated IOP in vivo. Although direct (gene by gene) comparison with previous studies mentioned earlier is not appropriate because of differences in the species, differences in the doses and type of steroids, and differences in the experimental setup (in vitro versus in vivo), the role of certain cell processes, pathways, or components is further corroborated (e.g., the role of ECM modulation).
In contrast to humans and monkeys, where the elevation of IOP induced by corticosteroids appears to occur in only approximately 30%
42 and 50%,
43 respectively, 100% of cows tested
5 and other ruminants
44 develop IOP elevation. Thus, these animals are ideal for studying the molecular mechanisms that lead to steroid-induced IOP elevation. The animal model that was used in our present work shares significant similarities with the human eye, both anatomically and in terms of the physiology of the aqueous humor formation. Bovine aqueous humor, as in humans, has a higher concentration of chloride than that of plasma,
45 and the isolated bovine ciliary epithelium transports chloride and is inhibited by carbonic anhydrase inhibitors.
46 On the outflow side, TM in the bovine eye is anatomically similar to the human TM (although a more formed pectinate ligament is present) and also shares other important homologies.
47 –49 The bovine aqueous plexus is the equivalent of the human Schlemm's canal.
50 At the same time it should be pointed out that there are also differences between the bovine and human TM function. In perfused anterior segments, human eyes do not show the phenomenon of “washout,”
51 whereas bovine eyes do.
52 It has been proposed that the human eyes have a more extensive network of elastic fibers in the cribriform plexus, preventing separation of inner wall from the juxtacanalicular tissue during perfusion.
53 This anatomic difference may account for the large amounts of extracellular material accumulating in this region in the cow eyes after relatively short term exposure to steroids.
6
We have previously reported the use of cows for studying the microscopic and ultramicroscopic changes at the level of the TM after steroid-induced IOP elevation.
6 In those experiments, as in the present study, we used animals that were subjected to at least 6 weeks of steroid treatment. Although investigating an earlier time point could detect a gene set enriched in genes leading to IOP elevation, it was elected to use the same time point as that in our earlier study. This time point was chosen not only to make the results comparable but also to ensure that IOP elevation would be present in all treated eyes, thus minimizing the variability inherent in experiments that do not use genetically inbred strains of animals (IOP elevation is not entirely synchronous, although it occurs in all animals tested). At the same time we did not want to have IOP elevated for prolonged periods of time to minimize secondary changes in gene expression. As expected, IOP elevations were achieved in all experimental eyes after approximately 4 weeks of treatment. IOP elevation was comparable in this set of animals to that in our previous report.
6
We have used oligonucleotide microarrays to study global gene expression in the TM. Oligonucleotide arrays have the advantage of greater specificity because they can be tailored to minimize chances of cross-hybridization.
54 Other major advantages of this approach include a uniform probe length and the ability to discern splice variants. The availability of genetic information for the cow has allowed us to use species-specific arrays, thus decreasing the ambiguity in gene calls. Using this approach we were able to identify a significant number of genes with changing expression as a result of steroid treatment and subsequent IOP elevation. The genes with changing expression can be in either one of the following four categories:
-
Genes that are involved in IOP elevation
-
Genes that are directly affected by steroid treatment but are unrelated to IOP elevation
-
Genes with expression that is changing because of IOP elevation
-
Genes with expression that is indirectly changing as a response to steroid-induced changes in the tissue but that are unrelated to IOP elevation
Thus gene expression changes can be either the direct effect of steroid therapy on TM cells (some causing IOP elevation) or occur secondarily as a result of activation/or suppression of other genes. The design of the present experiment does not allow us to determine which category the identified genes fall into, but does narrow the field of possible genes involved. Based on human genomic data, approximately half of the genes identified are potentially directly regulated by steroids. Indeed, some of the genes identified using the present approach have been previously implicated by others in the pathogenesis of steroid-induced IOP elevation and in open-angle glaucoma. For example, 7 (17.5%) of the 40 genes proposed to constitute the molecular signature of human glaucomatous TM
35 were also detected to be changing by the present analysis (these are aB-crystallin, cadherin, insulin-like growth factor–binding proteins, metallothioneins, thrombomudulin, transgelin, and tropomyosin).
Using microarrays to identify genes, of course, poses some limitations that are inherent to this methodology.
55,56 Genes with low-fold change are less likely to be detected than genes with dramatically different gene expression. In addition, a portion of the genes identified are false positives and some genes with truly changing expression fail to be identified. Thus, for example, myocilin whose expression is known to change in steroid-induced IOP elevation in perfused human
57 organ cultures failed to reach threshold values and was called “nonchanging.” Although this is rather surprising, it could also represent a true finding. Myocilin polymorphisms have not been associated with steroid-induced glaucoma in humans or primates
43 and myocilin induction by steroids has to our knowledge been reported only in cultured bovine TM cells
48 (but not in perfused bovine anterior segments).
To overcome some of the inherent limitations of microarrays, traditional strategies such as confirmation of gene changes using QRT-PCR can be used. It is generally accepted that QRT-PCR is more reliable than MA when it comes to individual genes.
58 However, QRT-PCR analysis can be performed on only a limited number of genes. We have used QRT-PCR to confirm a limited number of interesting genes. Some of these genes have been previously implicated in IOP elevation, whereas the role of others is yet unclear. In particular we would point out the changes in expression of
TYRP1. Lack of
TYRP1 has been implicated in glaucoma development in DBA/2 mice.
59
Using a bioinformatics analysis approach, we have identified a number of gene networks that are affected in steroid-induced IOP elevation in the cow TM. Some of these networks (
Fig. 4) are predictable based on what we already know about glaucoma.
TGFb is known to be involved in the pathogenesis of the disease.
60 Thus, even if the selection criteria for identifying genes with changing gene expression failed to detect change in
TGFb expression, its known role in the development of IOP elevation confirms the importance of the associated molecules within this network. Moreover, given the substantial number of genes in each of the networks identified one can safely assume that the whole network activity has been altered. One of the genes identified in this manner is
MAPK14 (mitogen-activated protein kinase 14, p38 MAPK) (see
Fig. 4). Its position in the network allows it to regulate a number of other genes, some of which have been implicated in glaucoma by the present work and that of others.
60 –66 MAPK14 is normally activated by various environmental stresses and proinflammatory cytokines and plays a critical role in the production of some cytokines (e.g., IL-6). In the bovine steroid-induced IOP elevation model
MAPK14 expression in the TM was upregulated 1.4-fold in the steroid-treated eye. It has been reported
67 that in open-angle glaucoma
MAPK14 is relatively unresponsive to IL-1 signaling, indicating a constitutive activation. Interestingly other MAPKs (e.g.,
MAPK10) seem to be downregulated in the bovine steroid-induced IOP elevation model.
Other networks, like the one depicted in
Figure 5, suggest that additional genes may be involved. Some of these genes such as
VEGF can be potentially interesting. Although it is unclear what its role in the TM is, one can speculate that since
VEGF can affect vascular permeability, it may have a similar role in affecting TM permeability. Acute elevation of IOP in perfused TM leads to
VEGF upregulation, although perfusion for longer times has been associated with downregulation of the gene.
35 Similarly, changes in expression of the adrenergic receptor beta 2 (
ADRB2), may be relevant because its main ligand (epinephrine) is known to affect outflow facility
68 through beta adrenergic receptors,
69 probably by increasing paracellular flow.
70 It is of course because of the design of these experiments that it is difficult to distinguish which of the steroid effects are related to IOP elevation and which are concurrent but unrelated effects of steroids on the TM. However, these networks provide a framework for confirming that individual genes are involved in a process and exploring how related genes are also affected. Moreover, it potentially allows identification of key molecules that can potentially affect the behavior of the whole network.
We have previously identified collagen VI as one of the components of plaques accumulating in steroid-induced glaucoma.
6 Given the presence of plaques in the extracellular space at the same time point in the same model, we further focused on changes in expression of genes encoding structural proteins of the ECM. Although collagen VI upregulation is detected by MA, collagen XXIV appears to have the greatest fold change among the collagen genes (approximately sevenfold upregulation). COL24A1 (Collagen XXIV A1) encodes one of the lesser studied fibrillar collagens. Its function is currently unclear, although based on the temporal pattern of its expression it has been suggested that it may participate in regulating type I collagen fibrillogenesis at specific anatomic locations during fetal development.
71 It has been previously detected in the bone, the cornea, and the retina. In addition (and most interestingly) it has multiple glycine–isoleucine pairs that are the target of proteolysis by MMP-1 (upregulation of which we have shown to be effective in preventing as well as reversing IOP elevation in the steroid-induced ovine model
72 ). In addition, collagen XII was downregulated. Collagen XII is a member of the FACIT (fibril-associated collagens with interrupted triple helices) collagen family. It is a homotrimer found in association with type I collagen, an association that is thought to modify the interactions between collagen I fibrils and the surrounding matrix. Other genes encoding for structural proteins that have been identified include laminin γ1, gelsolin, hyaluronan, and proteoglycan linking protein 1, latent transforming growth factor binding protein 2, and cadherin. It thus appears that even at this relatively early point after the induction of IOP elevation by steroids, significant change in the ECM of the TM occurs. This change is coupled with changes in the integrin pathway that allow cells to communicate with and control the composition of the ECM (see
Table 4).
Changes in gene expression do not by themselves affect cellular physiology unless they are followed by changes in protein production. Confirmation of the changes in protein amounts of some of the genes differentially expressed has traditionally been part of many studies of differential gene expression and has been done by immunoblotting. The very limited amount of protein that can often be extracted from small fragments of tissue make the task of confirming differential protein expression one at a time virtually impossible. In this study we have elected to confirm changes in the amounts of two proteins with commercially available antibodies that are documented to cross-react with the bovine protein. One of the proteins, GPNMB, is interesting because its absence is known to be critical for the development of glaucoma in DBA/2 mice similarly to TYRP1 protein as discussed earlier.
59 As expected in the steroid-induced cow model GPNMB is also downregulated.
In summary we have identified a number of genes with changing gene expression in the TM of the bovine steroid-induced IOP elevation model. A number of these genes are involved in the pathogenesis of this condition. It is hoped that understanding the interrelations of these genes and the sequence of molecular events that lead to IOP elevation will enable us to devise novel therapeutic strategies to treat steroid-induced glaucoma as well as other open-angle glaucomas. This understanding will be further enhanced from studying earlier points in the process of IOP elevation that we are currently pursuing.
Supported in part by National Eye Institute Grants R01 EY20670 and R03 EY16050, and an unrestricted grant from Research to Prevent Blindness (New York, NY).