We have tested the hypothesis, in vitro, that the TMEs release factors into the media, and that these factors upon binding to SCEs increase the permeability of the cell barrier formed by SCEs. If the same mechanism is operative in vivo, by implication, increasing transendothelial fluid flow across the SCE barrier is tantamount to facilitating aqueous outflow, as these cells form the last barrier crossed by this fluid upon exiting from the eye. The validity of our hypothesis is strongly supported by the evidence provided in our studies. The TMEs, which are activated by using a lasering procedure, are capable of releasing a large number of factors into the media. When aliquots of these factors are collected from the medium conditioned by the TMEs and added to SCEs, the putative factors activate the SCEs. That is, the SCEs exposed to this medium respond by producing many DE genes. Moreover, as predicted, the addition of media conditioned by the laser-activated TMEs to monolayers of untreated SCEs results in a 400% increase in SCE conductivity. The role of media factors is supported by control studies showing that boiling, diluting, or using medium from untreated TMEs abrogates the TME medium effects on SCE permeability. Finally, in two sets of experiments, we directly addressed the issue of the involvement of media-borne molecular factors released by TMEs. In one set of experiments, we demonstrated that the TMEs do synthesize and release IL-8 by demonstrating that its gene is up-regulated and that the corresponding mRNAs undergo a congruous induction, which results in the synthesis of the corresponding IL-8 protein. The second set of experiments demonstrates, using ELISA, that three other cytokines are released into the media by TMEs and, most important, when each of the four candidate cytokines are added individually to SCEs, the conductivity increases in agreement with our hypothesis.
An unexpected discovery is that the responses to the laser treatment are cell-specific, because lasering TMEs yields the differential expression of 1,570 genes compared with lasering SCEs, which yields only 40 DE genes. These differential gene responses are correlated with the potency effects of medium conditioned by each cell type. Medium conditioned by the highly laser-activated TMEs induces correspondingly large responses when added to either untreated TMEs or SCEs, generating 829 and 1,120 DE genes, respectively, for a total of 1,949. On the other hand, medium conditioned by the weakly laser-activated SCEs induces congruous small responses when added to either SCEs or TMEs, producing 328 and 12 DE genes, respectively, for a total of 340. Thus, there is at least a 500% greater induction of DE genes by TME-conditioned medium compared with SCE-conditioned medium (ie, 1,949 versus 340 DE genes). These gene differences are reflected as well in the conductivity increases induced by each cell type. Medium conditioned by the lasered TMEs induces a 200% greater increase in conductivity when added to TMEs and SCEs, compared with medium conditioned by the lasered SCEs when added to the same two cell types.
Another important finding is that the cell-to-cell interactions proceed in both directions, involving TME-to-SCE and SCE-to-TME relationships, as well as mutual exchanges involving TME-to-TME and SCE-to-SCE associations. In the case of some of these interactions, the conductivity effects are not strictly related to the differential induction of genes. For instance, the lasered SCEs undergo the DE of only 40 genes, but medium conditioned by these lasered SCEs when added to naïve SCEs induces 320 genes, a response that represents an eightfold increment. To explain these seemingly disparate effects, we propose that SCEs have a receptor profile and binding affinity that favors factors released by lasered SCEs. Other responses proceed in the opposite direction, such as those when medium conditioned by lasered SCEs is added to naïve TMEs (12 DE genes). The SCEs undergo the induction of 320 genes when exposed to SCE-cm, which suggests that this medium probably contains a large complement of factors. Yet, when the SCE-cm is added to naïve TMEs, these cells respond by undergoing the DE of only 12 genes, the smallest response detected. In this circumstance, we conjecture that the binding affinity and receptor profile of TMEs is particularly insensitive to the effect of factors released by the SCEs. In future experiments, we will control for differences in cell activation by manipulating the content of chromophores given, as well as the amount of laser irradiation delivered to each cell type, so that the desired response intensity is achieved. Understanding these differences in “sensitivity” is an important consideration in choosing factors to induce conductivity increases affecting one cell type in preference to the other for the potential treatment of glaucoma.
The finding that cytokines released by TMEs regulate the permeability of the SCE barrier is novel and to our knowledge has not been previously proposed. How this TME mechanism contributes to the two functions of the trabecular meshwork to facilitate aqueous outflow and prevent the reflux of blood is not readily apparent. The requirements of such a mechanism are highlighted when one considers how the SCEs help the trabecular meshwork carry out both of these functions. Here we are referring to the formation of giant vacuoles by the SCEs. When the IOP level exceeds the episcleral venous pressure, the pressure along the basal cell surface exceeds that in the apical surface of the SCEs and provides the driving force to induce the formation of giant vacuoles. By providing the SCE barrier with an accessory outflow route, these vacuoles in turn promote the more rapid egress of aqueous. When the episcleral venous pressure exceeds the IOP, the pressure is now higher along the apical than it is along the basal cell surface, eliminating the driving force that induces the formation of giant vacuoles. Thus, giant vacuoles are no longer formed, and the barrier becomes more resistant, as would be required to prevent the reflux of blood. Measurements of the conductivity of monolayers of cultured human SCEs in vitro yield data that are consistent with these concepts. For instance, when perfused with fluid flowing in the same direction as followed when aqueous exits from the eye (ie, outflow direction), the conductivity of the SCEs measures 5.23 μL/min/mm Hg/cm2
, as the endothelium behaves as a leaky monolayer that can easily promote the outflow of aqueous. When the SCE monolayer is perfused in the opposite, or reflux, direction, the conductivity measures 0.66 μL/min/mm Hg/cm2
, or eight times more resistant. Such a resistance would be required to prevent, to some extent, the reflux of blood.2
Therefore, these considerations help us understand how, by linking the giant vacuole formation process with that of the relationship between the IOP and the venous pressure in the lumen of Schlemm’s canal, the SCEs function to assist the trabecular meshwork in carrying out its dual functions of facilitating aqueous outflow and preventing the reflux of blood.
We now use these concepts to propose that a mechanism exists for the TMEs that is essentially similar to that described for the SCEs. The trabecular meshwork and the lining TMEs, as well as Schlemm’s canal and the lining SCEs, undergo deformation and stretching with changes in intraocular pressure.30
Moreover, stretching the TMEs by mechanical means or by increasing the intraocular pressure elicits a wide variety of important biochemical responses.31–34
Assuming that the TMEs have stretch receptors, when the IOP is greater than the venous pressure, the increased tension makes the trabecular beams and cords taut, thus triggering the stretch receptors to activate the TMEs to release vasoactive factors that then will increase flow across the SCEs. When the IOP is less than the venous pressure, the beams and cords become flaccid, resulting in the opposite response, which should increase the resistance presented by the SCEs so as to resist the reflux of blood. If such a tension-sensitive mechanism does in fact exist, it is likely that miotics, like pilocarpine, by inducing the contraction of the ciliary muscle and increasing the tension along the trabecular meshwork beams and cords, could also activate the stretch receptor to turn on the TMEs. These cells, under the influence of the miotic-mediated increase in tension, would then release the factors required to increase the SCE conductivity, and thus the egress of aqueous. We propose that the mechanical effects generating tension and biologic mechanisms releasing vasoactive factors work together. These interactions account for the action of miotics during glaucoma therapy, instead of being due to strictly mechanical effects as traditionally credited for the action of this drug.35
Importantly, the validity of this theory can be readily tested using the gene activation approaches described in the present paper along with the use of TMEs grown over stretchable silicone sheets.
In primary open-angle glaucoma (POAG), the population of trabecular meshwork endothelial cells is markedly decreased compared with that of age-matched healthy subjects.36–40
This progressive decline in cell density results in the loss of 0.58% of the total number of cells per year and is most pronounced in the inner layers of the filtration zone of the trabecular meshwork. The inner trabecular cells, which are the first encountered by aqueous humor, may be prone to injury by free oxygen radicals carried in this fluid.39
When we first noticed the loss of TMEs in POAG, it was difficult to comprehend how this cell loss could have a negative impact on the facility of aqueous outflow and the pathogenesis of glaucoma,36
particularly in view of the generally held concept that the greatest resistance to aqueous outflow is presented by the SCEs.3
Recently, sophisticated assays have been carried out which demonstrate that there is extensive oxidative DNA damage involving the trabecular cells of patients with POAG, affecting the filtration zone and the inner trabecular meshwork layers.41
Additionally, we are interested in understanding the particular mechanisms involved in the loss of trabecular meshwork cells. Other studies report that incorporating a particular type of myocilin mutant known to be present in vivo in certain types of open-angle glaucoma into TMEs in vitro (ie, Pro370Leu) actually results in what is referred to as “killing” of the cultured human TMEs. The TMEs’ demise is due to misfolding, aggregation, and buildup of this protein in the endoplasmic reticulum of the trabecular cells.42
Whether due to oxidative DNA damage or the abnormal processing of protein folding (ie, proteomics) by the TMEs in POAG, there is increasing evidence suggesting the involvement of several mechanisms whereby the normal population of trabecular meshwork cells is affected by dysfunction and death. In view of the present study, it is now becoming increasingly clear how such a loss of trabecular meshwork cells, by reducing the quantity of cytokines released by a diminished population of TMEs, could have a negative impact on the homeostasis of aqueous outflow. The reduced load of cytokines and other factors may not maintain the porosity of SCEs necessary to facilitate aqueous outflow, and the IOP may rise to the abnormal levels characteristic of many patients with glaucoma.
This study could not have been accomplished without the use of the F-D Nd:YAG laser, because this instrument, when applied using low-fluence light energy, preserves the baseline permeability properties of the lasered cells. Our results provide a new understanding of the mechanism of action of this novel glaucoma laser therapy based on the activities of the TMEs and SCEs, instead of purely mechanical effects.9,43
In view of the fact that lasering effects appear to be cell-specific, we are inclined to support a more prominent role for the TMEs, which are most intensely activated by F-D Nd:YAG laser treatment. In addition, it is important to recall that the TMEs also release matrix metalloproteinases, which in promoting fluid flow across the extracellular matrix, also participate in facilitating the overall rate of aqueous outflow.10
It is also important to note that the interactions between TMEs and SCEs proceed in both directions and involve relationships within cells of a given type. This is important for several reasons. Interactions among TMEs allow for these cells to release factors that could affect the TMEs lining the outermost aqueous channels, which must be crossed before aqueous can pass into the juxtacanalicular tissues. The paracellular route of the outer TMEs is more porous than that in other TMEs lining the innermost trabecular meshwork beams and cords. Perhaps this particular widening of the paracellular route of TMEs lining the outermost trabecular meshwork is related to the cumulative effects of cytokines released by the entire population of TMEs becoming most concentrated, and having the greatest effect in TMEs near the juxtacanalicular tissues. Similarly, although the SCEs are less activated, factors released by these cells are particularly potent in promoting transendothelial flow across SCEs, as demonstrated by our experiments. Thus, the activation of SCEs by the laser treatment may be particularly effective in promoting transendothelial flow across the SCE barrier.
We conclude by noting that the use of the F-D Nd:YAG laser, and the in vitro methods described, has already allowed us to identify four cytokines released by lasered TMEs. Completion of this survey by identifying cytokines and chemokines involved, among the known 298 cytokines and chemokines, is a realistic goal, and such knowledge may enhance our future ability to manipulate aqueous outflow using some of these factors in the treatment of glaucoma.