Our experiments have provided substantial support for the hypothesis that TMEs release factors into the media, and that these factors upon binding to SCEs increase the permeability of the SCE barrier. SCEs form the last cellular barrier traversed by aqueous as it exits from the eye and enters into the venous circulation. As such the SCE barrier is strategically located to act as a “control” site, so that increasing the permeability of this barrier augments the egress of aqueous from the eye. TMEs, according to our findings, drive a mechanism controlling the SCE permeability by releasing vasoactive cytokines and other factors that have the capacity to increase the permeability of the SCE barrier. In fact, the addition of media conditioned by the irradiated TMEs to monolayers of untreated SCEs results in a 400% increase in SCE conductivity. The involvement of media borne factors secreted by TMEs has been addressed directly in several ways. In one set of experiments we demonstrated that in irradiated TMEs the gene for IL-8 is upregulated, and that the corresponding mRNAs undergo a congruous induction, resulting in the synthesis of the IL-8 protein. In another set of experiments, we demonstrated that three other cytokines are released into the media by TMEs as a function of the number of laser pulses applied and the power used. When each of these four cytokines is added individually to SCEs, the conductivity increased as we have postulated. The role of media factors is also supported by control studies showing that boiling, diluting, or using medium from untreated TMEs, abrogates the TME medium effects on SCE permeability.
Our findings highlight the importance of the well differentiated endothelial cells of the trabecular meshwork and Schlemm’s canal as a system in which to study cell-cell interactions. These interactions are complex and proceed in both directions, involving TME-SCE and SCE-TME relations, as well as mutual exchanges concerned with TME-TME and SCE-SCE associations. For example, we have learned that the responses to light irradiation treatment are specific for each cell type, with irradiated TMEs undergoing a DE of 1570 genes compared to irradiated SCEs, which undergo a DE of only 40 genes. Similarly, the responses to the addition of media conditioned by irradiated cells are specific to each cell type. Treatment by the addition of media conditioned by irradiated TMEs to either untreated TMEs or SCEs induces the DE of 829 and 1120 genes respectively (total of 1949). In comparison, medium conditioned by laser activated SCEs when added to either SCEs or TMEs induces only 328 and 12 DE genes (total of 340). Thus, there is at least a 500% greater induction of DE genes by TME conditioned medium compared to SCE conditioned medium. These differences are reflected as well by the conductivity increases induced by each cell type: medium conditioned by the lasered TMEs induces a 200% greater increase in conductivity compared to medium conditioned by the lasered SCEs when added to TMEs and SCEs.
The finding that cytokines released by TMEs regulate the permeability of the SCE barrier is novel. It is not readily apparent how this TME driven mechanism contributes to the two functions of the conventional aqueous outflow pathway facilitating aqueous outflow and preventing the reflux of blood. We propose that a mechanism exists for TMEs, which is essentially similar to that described in the introduction driving the formation of giant vacuoles by 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.21
Stretching TMEs by mechanical means or by increasing the IOP elicits a wide variety of important biochemical responses.22–25
Assuming that TMEs have stretch receptors, the increased tension makes the trabecular beams and cords taut, thus triggering the stretch receptors to activate TMEs to release vasoactive factors that will increase flow across SCEs. When the IOP is less than the venous pressure, the beams and cords become flaccid, thus increasing the resistance presented by SCEs.
If such a tension sensitive mechanism does in fact exist, it could provide a biological basis for the action of miotics during glaucoma therapy. For example, miotics like pilocarpine, upon inducing contraction of the ciliary muscle, also increase tension along the trabecular meshwork beams and cords, which in turn activates the stretch receptor to turn on the TMEs. TMEs, under the influence of the miotic mediated increase in tension, would then release the factors required to increase SCE conductivity, and thus the egress of aqueous. We propose that the interaction of mechanical effects generating tension and biological mechanisms releasing vasoactive factors work together during the miotic mediated increase in aqueous outflow, and perhaps even during accommodation.
In primary open angle glaucoma (POAG), trabecular meshwork endothelial cells are markedly decreased relative to those of age matched normals.26–30
This progressive decline in cell density results in a loss of ~0.58% cells/year, which is most pronounced in the inner layers of the trabecular meshwork’s filtration zone. The inner trabecular cells may be prone to injury by free oxygen radicals carried in the aqueous outflow.29
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,26
particularly in view of the generally held concept that the greatest resistance to aqueous outflow is presented by the SCEs.31
Recent evidence demonstrates that there is extensive oxidative DNA damage involving the trabecular cells of patients with POAG, affecting the filtration zone and inner trabecular meshwork layers.32
Other studies report that incorporating a specific type of myocilin mutant known to be present in vivo in certain types of open angle glaucoma into TMEs in vitro (that is, Pro370Leu) results in “killing” of cultured human TMEs.33
The demise of TMEs is the result of misfolding, aggregation, and build up of this protein in the endoplasmic reticulum of trabecular cells.33
Whether caused by oxidative DNA damage or abnormal processing of protein folding by TMEs in POAG, there is significant evidence that the normal population of trabecular meshwork cells is affected by dysfunction and death. In view of our findings, it is 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.
Our results provide a new understanding of the mechanism of action of glaucoma laser therapy using the F-D N:Y laser, based on the activities of TMEs and SCEs.34,35
In view of the fact that lasering effects appear to be cell specific, we suggest a more prominent role for TMEs, which are most intensely activated by F-D N:Y laser treatment. The laser treatment is proposed to function by intensely activating the TMEs, which release cytokines that flow downstream and bind to SCEs. The SCEs, by undergoing a decrease in resistance, allow the more rapid egress of aqueous from the eye. It is important to recall that TMEs release matrix metalloproteinases, which by promoting fluid flow across the extracellular matrix, also participate in facilitating the overall rate of aqueous outflow.36
The interactions between TMEs and SCEs proceed in both directions, and involve relations within cells of a given type. Interactions between TMEs allow for these cells to release factors that would 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 of TMEs in other regions. Perhaps this 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. Although SCEs are less activated, factors released by these cells are particularly potent in promoting transendothelial flow across SCEs, as demonstrated by our experiments. We conclude that the use of the F-D Nd:Y laser, and the in vitro methods described, has allowed us to identify four cytokines released by lasered TMEs. Completion of this survey of the known 298 cytokines/chemokines is a realistic goal and such knowledge may enhance our future ability to manipulate aqueous outflow during glaucoma therapy.