Ординатура / Офтальмология / Английские материалы / Mechanisms of the Glaucomas_Shields, Tombran-Tink, Barnstable_2008

could also affect outflow facility by adjusting cell–matrix interaction or viscoelastic properties of the tissue.

In addition to the above genes representing novel processes, a very well-established trabecular meshwork gene was up-regulated. Matrix metalloproteinase 2 (MMP2) is a collagen IV cleavage enzyme which along with its inhibitor TIMP2 has long been associated with trabecular meshwork ECM turnover (89,90). As such, it is a key enzyme for maintenance of outflow facility. Transcription of MMP2 is up-regulated with pressure in this human system (15), and translation and activity of the enzyme are increased in human, porcine, and bovine trabecular meshwork cells after subjecting them to 72 h of different mechanical strain models (21,91,92). Although some of the mechanical insults showed no change in MMP2 mRNA levels (93), the combined overall evidence from different laboratories, being at the transcriptional, translation, or enzyme activation level, demonstrated a role for MMP2 in response to insult during homeostasis.

Lastly, the presence of three other interesting genes reflects a potential involvement of a protein degradation mechanism during mechanical stress. One of them is the induction of the proteosome 26S subunit (PSMD4), a multicatalytic endopeptidase that plays a major role in protein degradation (65). The other two are chaperonincontaining TCP1 and protein disulfide isomerase, which are involved in protein folding

(70,75). The chaperonin-containing TCP1 complex assists in the folding of newly translated polypeptide substrates, which include among others the cytoskeletal proteins actin and tubulin (94), two proteins that have long been associated as modulators of outflow facility (95). Protein disulfide isomerase catalyzes thiol interchange reactions that involve oxidation, reduction, and isomerization of disulfide bonds (75). It is an intrinsic component of the protein folding mechanism. Deformation and unfolding of proteins occurs under mechanical stretch, and it is part of important processes in living systems, such as elasticity and cell adhesion (96). Under a stretching force, polypeptides unfold and subsequently refold (96). Proteosomes protect cells from stress by degrading unfolded proteins and thus remove the damaged polypeptides from the cell environment. Therefore, it is not surprising that a protein folding and cleaning up mechanism would be part of an overall protective response of the trabecular meshwork to pressure spikes.

Human Genes Expressed During the Pre-Homeostatic Period

The response to pressure using human perfused system (see Fig. 4) showed that 1 and 6 h of insult appear to show the beginning of a homeostatic response. In Fig. 6, we have gathered a selection of the most up-regulated human genes obtained during that period (97–114). The results were obtained in our laboratory by the use of macroarrays (14) and DNA chips (unpublished). Looking overall at the categories of the functions encoding by these genes, tells us that in most cases those functions are associated with mechanisms that each in their own way would tend to facilitate aqueous humor outflow.

A fairly good proportion of genes were involved in cytoskeletal reorganization and consequently, in changes of cell shape (transgelin, cornifin, spectrin, and tubulin) (99,100,104,111). It is well-established that the cytoskeleton plays a role in the

1 hour: Borrás (unpublished)

6 hours: From data on Gonzalez et al. IOVS 41, 352 (2000)

Fig. 6. Selected Human Genes Modified During Pressure-Induced Pre-Homeostasis Encoding Functions Potentially Relevant to TM Physiology.

regulation of pressure and that agents that disrupt the cytoskeleton increase outflow facility in living animals, perfused enucleated eyes, and anterior segments (95). Another identified function is that of stress defense, which would be represented by genes encoding metallothioneins, B-crystallin, and superoxide dismutase and could be an indication of the importance of regulating the trabecular meshwork redox state (112–115). Metallothioneins are heavy metal binding proteins which in other tissues are known to play important roles in defending cells against the cytotoxicity of heavy metals (zinc and cadmium) and oxidative stress (113). Redox-sensitive genes have been shown to be mechanosensitive in human vascular endothelial cells subjected to shear stress (116). A signaling function would be represented by the activin receptor, vascular endothelium growth factor, interleukin 6, and substance P precursor (97,98,106,107) genes. These molecules could signal temporary vascular permeability which in turn could facilitate the passing of the aqueous humor through the outflow tissue. In recent studies, transcription and secretion of IL6 was induced by cyclic mechanical stress (117), and its secretion was also increased after 48 h of hydrostatic pressure in microglia

but it was reduced in astrocytes (118). Furthermore, this study showed that survival of RGCs under hydrostatic pressure was enhanced by using microglia-conditioned medium but reduced using the astrocyte-conditioned one. Addition of recombinant IL6 to the RGC under pressure cultures, does also promote survival (118). The authors proposed that IL6 has a neuroprotectant effect on RGCs.

The induction of secretogranin II (108) could represent activation of the secretory pathway, and this enhancement could influence the release of other ECM proteinases, such as MMP3. Transcription of MMP3 is also affected and could contribute to the reorganization of the trabecular meshwork ECM (109,110).

Porcine Genes Responding to Mechanical Stress in a Homeostatic Manner

One way by which the anterior segment of the eye controls the resistance to the flow of aqueous humor and creates a physiological pressure is by stretching the whole trabecular meshwork tissue. Under conditions of higher pressure exerted across the outflow pathway, the trabecular meshwork undergoes different degrees of stretching. This stretching response is associated with a feed back, homeostatic mechanism that attempts to correct and reduce the resistance (21). A well-controlled high-throughput study searching for genes whose expression is altered during the exertion of stretching has recently been conducted (93). The study used early passage primary trabecular meshwork porcine cells and the glass bead mechanical distortion model described earlier. The porcine RNA, isolated at three different time periods (12,24, and 48h) of stretching, was hybridized to microarray chips containing human cDNA clones. The advantage of using cDNA rather than oligonucleotide chips in this case is that it allows the hybridization of DNA from two different species with minimal interference of mismatching sequences.

Using stringent criteria, of the 8500 genes in the DNA chips, a total of 126 transcripts were up-regulated and 29 down-regulated. This represented a similar pattern than the one obtained in human eyes during the pressure homeostatic response (15). Of the up-regulated genes, 27 encoded ECM proteins (21%), 20 encoded proteins involved in transcription/translation (16%), 37 encoded stress/metabolism (29%), and 42 (33%) encoded proteins mostly involved in cytoskeletal functions. Only 13 genes were altered at the three time periods while the remaining 113 were mostly altered at one of three time points. This narrow window of induction (e.g. 12 or 24 h) is an indication of a rapid sensing and response mechanism on the part of the trabecular meshwork cells to mechanical distortions. It is also noticeable that while most altered genes change values range between 0.5- and 3-fold, the genes encoding metallothioneins (five isoforms in the chip) changed approximately by 30to 100-fold. Nine isoforms of metallothioneins are also among the highest induced by a 6-h elevated pressure in the human perfused system (14). It is interesting to think why these proteins are so strongly affected under the two systems such finding validates cellular stretch of the trabecular meshwork cells as a direct consequence of elevated pressure and, raises the intriguing possibility that these mechanical stresses might provoke a toxic accumulation of zinc. In the brain system, some injuries, like blunt head trauma and ischemia induce intracellular increases and releases of zinc that lead to zinc dyshomeostasis and neuronal cell loss

(115,119).

Using the stretch boat model in primary human cells, investigators found that myocilin expression was up-regulated under stretch conditions (22). A dramatic increase of myocilin was also reported using cells of porcine origin undergoing stretch using the glass bead system (93).

Optineurin, previously identified as FIP2 and NRP (158,159), is a protein induced by TNF that can block anti-apoptotic activity (158). Studies form our laboratory showed that optineurin was up-regulated in perfused human anterior segments by pressure but with a slightly different pattern than that of myocilin. As it occurred with myocilin, no significant up-regulation was observed at earlier times. However, significant upregulation was observed after a period of 2 days, which increased dramatically by 7 days (55). A different study using the same system but shortening the insult to 24 h did not observe changes in optineurin expression response to pressure elevation (153). Interestingly, what appears in the titles of both manuscripts as contradictory findings do actually agree in content once duration of insult is considered. No studies are yet available on the response to pressure of the WDR36, a recently identified gene involved in T-cell activation and co-regulated with IL-2 (160). Although optineurin and WDR36 were present in the chip of the stretch studies, the expression of both genes remained unchanged (93).

Effects of Pressure on Other Single Genes

Human Neuronal Olfactomedin A

When myocilin was discovered, the best fit of the new sequence using a Blast search occurred between the C-terminus of myocilin and members of the olfactomedin protein family, in particular with neuronal olfactomedin (161). Proteins of the olfactomedin family are known to form oligomers, and these interactions occur through essential cysteine residues that happen to be conserved in myocilin (162). Also, a great majority of myocilin mutations associated with glaucoma have been mapped to the olfactomedin domain (163). Taken these facts together has led to the proposal that formation of myocilin protein heterodimers through this domain might be relevant for the development of the disease (164). Because of the induction of myocilin by pressure, a study in our laboratory was conducted to investigate whether there was a potential co-regulation of human neuronal olfactomedin A and myocilin. It was interesting to find that in the same individual, both proteins responded to pressure with the same trend. That is, their expression was induced after longer high IOP insults (see Fig. 11). Although this co-induction is not a proof of protein binding or a relation between the two proteins, the result is nevertheless intriguing.

Human Chloride Channels

As we mentioned earlier, we believe that the trabecular meshwork relies on more than one mechanism to regulate outflow resistance (14). One of those mechanisms might involve regulation of cell volume. It has been demonstrated that experiments that induce trabecular meshwork cell shrinkage result in an increase in outflow facility, whereas those inducing swelling reduce outflow (165,166). Chloride channels allow the diffusion of negatively charged ions and are involved in volume regulation (167). Two members of the voltage-gated chloride family, CLC-2 and CLC-3, have been

microarray studies are under more sensitive measurements identified as trabecular meshwork mechanosensitive responders.

COMPREHENSIVE SELECTION OF POTENTIALLY RELEVANT MECHANOSENSITIVE GENES

In Fig. 13, we have compiled genes which we have repeatedly found in the macro/microarrays of human trabecular meshwork as those most affected by elevated pressure. We have revised the current literature and cross-checked our selection with other reported mechanosensitive trabecular meshwork genes, especially those responding to mechanical stretch. Despite the different species and models used, we are beginning to see a number of similarities both on the individual genes and/or functions encoded by the affected genes.

In the ECM, it is intriguing to observe expression changes in a number of osteogenic genes. In addition to MGP, others like osteonectin, osteoblast-specific factor, osteoglycin, and osteomodulin are genes associated with the development of bone and cartilage, as well as vascular calcification (171). Although MGP conserves the inhibition of calcification role in the trabecular meshwork (86), the function of these additional osteogenic genes in the outflow tissue is not yet known. Mechanical stimuli play a role in bone formation and markers such as osteoglycin expression have been found to be regulated by mechanical stimuli such as tension, shear, or hydrostatic stress in pre-osteoblasts and chondrocytes (85,134,172).

It is interesting to note that some of the trabecular meshwork “classic” ECM genes such as tenascin, CTGF, fibronectin, and proteoglycans (173,174) are also mechanosensitive. Although the studies here just looked at transcriptional control, the mechanical response extends also to the domains of these ECM proteins. In other systems, the fibronectin type III domain, present in the matricellular proteins of Fig. 7, has been shown to contain a mechanosensitive control element at the RGD binding site (175), which could affect cell–matrix adhesion.

The original mechanosensitive nature of the gene precursor of substance P in the trabecular meshwork cell (14) has now been seen in our laboratory in more than one condition and under different experimental procedures. The fact that no neuronal bodies are present in the trabecular meshwork indicates that this tissue has not only the potential for producing substance P but of using this neuropeptide, which had been previously identified as IOP modifier (176), for autocrine IOP regulation.

Intracellularly, a number of stress protectors are responding to mechanical stimuli. Metallothioneins seemed to be overall the more responsive. Human B-crystallin, a small heat shock protein abundant in the lens, had already been identified as a mechanosensitive gene in the trabecular meshwork (23,24) and is now consistently showing in pressure and stretching arrays. Others, such as superoxide dismutase, primer protector of oxidative stress (114), are known to be activated by other stresses in the trabecular meshwork (177), appeared so far only in the human but not porcine microarray studies. One wonders whether this difference is a reflection of differences in IOP regulation between species (178).

The appearance of cytoskeletal-related genes, such as alpha tubulin and tropomodulin are not a surprise. Cytoskeletal changes affecting the shape and the intracellular scaffold