We were interested in how the TM cytoskeleton affects outflow. We had the idea that epinephrine, as first suggested by Dr. Bárány (Bárány, 1968
), by interacting with the cytoskeleton, could change the contractile properties of the TM. Bill and Bárány had shown that epinephrine increased outflow facility (Bárány, 1968
; Bill, 1969
). This resulted from a β2 adrenergic receptor mediated effect on the TM and an increase in cAMP (Neufeld et al., 1972
; Neufeld et al., 1973
; Neufeld and Sears, 1975
; Neufeld and Bartels, 1982
; Sears, 1966
; Sears and Neufeld, 1975
). Our disinsertion model showed that the iris and ciliary muscle were not involved in this outflow facility effect (Kaufman and Bárány, 1981
; Kaufman and Rentzhog, 1981
What was the physical mechanism? Old work in German anatomic literature (from 1938, citation unavailable) showed that epinephrine could change the shape of cat omentum cells. If epinephrine worked on facility by contracting the TM, could actin microfilament disruption relax the TM and block the effect?
Cytochalasins thus became interesting compounds to probe the question. Cytochalasins, which are fungal metabolites, interfere with the process by which globular cytoplasmic actin aggregates into actin microfilaments (Brown and Spudich, 1981
). First “control” experiments tested the hypothesis that cytochalasins had no effect on, or even reduced, outflow facility. Surprisingly, cytochalasins dramatically increased outflow facility, albeit briefly, following intracameral bolus injection in intact as well as ciliary muscle-disinserted eyes (Kaufman and Bárány, 1977
; Kaufman et al., 1977
). This result started our long hunt. The effect was associated in time with a very expanded TM () as a result of the fluid pressure being higher in the anterior chamber than in Schlemm’s canal and thus pushing its way through the juxtacanalicular region where the weakened cytoskeleton and cell attachments allowed tremendous expansion of intercellular spaces and washout of extracellular material (Svedbergh et al., 1978
). Also present were breaks in the inner wall of Schlemm’s canal. Aggregations of platelets were present trying to close the ruptures at the inner wall epithelium, as they would for any damaged blood vessel. Clearly cytochalasins were not agents one was going to use clinically but it did leave us in a conundrum as to what was going on.
Figure 2 Schlemm’s canal and cribriform meshwork approximately 30 min after intracameral infusion of 5μg of cytochalasin B. Inner wall endothelium (SC) demonstrates ruptures (arrow) and abnormally large invaginations (I). Extracellular material (more ...)
Meanwhile human TM (HTM) cells were isolated and cultured by Jon Polansky (Polansky et al., 1979
). Treatment of HTM and monkey TM cells in culture with cytochalasin B resulted in marked changes in cell shape associated with organizational changes in actin filaments (Ryder et al., 1988
; Weinreb et al., 1986
). Treatment of HTM cells grown on filters with cytochalasins resulted in increased hydraulic conductivity (Perkins et al., 1988
Cytochalasins also potentiated the outflow facility increasing effect of epinephrine (Robinson and Kaufman, 1991
). Intracameral doses of cytochalasin B and epinephrine, which were subthreshold for increasing outflow facility when given singly, produced significant outflow facility increases when given concurrently. Subthreshold doses of cytochalasin B given concurrently with a maximal dose of epinephrine, produced significantly larger outflow facility increases than a maximal dose of epinephrine alone.
More definitive proof that epinephrine and cytochalasin B both were acting through disruption of actin microfilaments was provided with additional experiments using phalloidin, which antagonizes cytochalsin B’s effects on actin in other biologic systems (Low et al., 1975
). Phalloidin itself did not affect outflow facility but it inhibited up to 50% of the outflow facility increasing effect of cytochalasin B and of epinephrine (Robinson and Kaufman, 1994
). Thus it was clear that actin filaments were involved in regulating aqueous outflow.
Already at this point the collaboration involved myself, Dr. Lütjen-Drecoll, Dr. Bill, Dr. Bárány, and Dr. Rohen, and thus was quite multinational.
The search for better cytoskeletal compounds and better mechanistic understanding began. Cell adhesions, unlike what you see under the microscope, are not static structures but rather are dynamic – they come and go, vary in tightness, location, adhesivity – depending on what is going on in the environment of the cell. They are very complex with many structural proteins and many housekeeping signal transduction proteins that govern the assembly and disassembly of the junctions and the interactions of the structural proteins. The complexes are tied to the actin cytoskeleton and functionally to acto-myosin contractility (Geiger et al., 2001
). In HTM cells this is visualized as long actin filaments, connected to vinculin providing structural anchoring to the plasma membrane at the junctional complexes, in turn linked to the matrix molecules on the other side (Tian et al., 2000
). One can manipulate these adhesions with hormones, drugs, or mediators, but the system also is governed by the amount of force applied to and by various components, by stretch, shear stress and other physical factors. For instance, stretch induced external force (e.g., intraocular pressure) may be counteracted by actin-myosin contractility force () (Geiger and Bershadsky, 2002
). This may be analogous to a reflex, and act as part of a complex regulatory system in a delicate balance depending on what the tissue is trying to do.
Figure 3 Focal Adhesion (FA) as a Mechanosensor. Focal adhesion is a multi-molecular complex connecting the extracellular matrix with the actin cytoskeleton. Heterodimeric transmembrane integrin receptors (red) bind matrix proteins via their extracellular domains, (more ...)
About this time, at the TM symposium in Chatham, MA in 1993. an encounter with Benny Geiger, the discoverer of vinculin and perhaps “the king of the cytoskeleton,” led to my sabbatical in his laboratory at the Weizmann Institute of Science in Rehovot, Israel in 1995–1996. He was working in all these areas, but not in the eye. I spent a year there amongst people of many different cultures and disciplines.
We began to understand the biochemical pathways involved that would allow us to regulate assembly and disassembly of the actin microfilaments. Myosin II-driven contractility plays a crucial role in the assembly and maintenance of two domains of the actin cytoskeleton, stress fibers and associated focal adhesions that link the actin cytoskeleton to the extracellular matrix. Formation of stress fibers and focal adhesions in the cell is triggered by the small GTPase Rho, which activates members of the Rho-associated kinase family (Burridge and Wennerberg, 2004
). Stimulating the Rho pathway and enhancing phosphorylation of the myosin light chain increases contractility. Conversely, inhibiting the Rho pathway and myosin light chain kinase with a variety of small molecules (H-7, ML-7, Y-27632) (Epstein et al., 1999
; Rao et al., 2001
; Tian et al., 1998
) or with bacterial toxins such as C3 (Liu et al., 2005
), will uncouple actin from myosin, resulting in relaxation of the cells and disassembly of the actin cytoskeleton. In addition to small molecules and toxins, there are other proteins, such as caldesmon, that uncouple the linkage of actin and myosin II resulting in focal adhesion disassembly and loss of actomyosin contractility (Helfman et al., 1999
We investigated in cells, how these substances worked and then were able to convey results, plans, course corrections, etc., within the same day, via email, phone calls and fax, back to my laboratory in Wisconsin where experiments were conducted in living nonhuman primates within days. It turned out that the dosages in cells were very predictive of what was going to happen to outflow facility in the live monkey.
One molecule that we examined originated from Latrunculia (now Negombata) magnifica, a sponge that lives in the bottom of the Red Sea. Latrunculins disrupt actin filaments in a much more gentle and perhaps physiologic way than cytochalasins; they inhibit the assembly of actin by binding free actin in the cell. Consequently, the actin microfilament degrades, resulting in loosening of cell-cell junctions, rounding of cells and cell separation. shows a bovine aortic endothelial (BAEC) cell stained for actin following treatment with 0.2μM latrunculin-A for 5 hours, resulting in attenuation of the actin filaments.
Bovine aortic endothelial (BAEC) cells stained for actin following treatment with 0.2μM latrunculin-A for 5 hours, resulting in attenuation of the actin filaments. [original]
Administration of latrunculin B into the anterior chamber of a monkey eye causes a dose-dependent increase in outflow facility (Peterson et al., 2000
). Further, infusing the drug into the anterior chamber, allowing the facility effect to develop, then washing the drug out with drug-free solution, and stopping the infusion for an hour reveals no loss of the facility effect when the drug-free infusion is restarted. However, leaving the system off for 4 hrs after the washout reveals that the outflow facility has returned to normal, but when the drug-free infusion is resumed the facility increases as it did during the initial drug infusion (Peterson et al., 1999
). Treating the normotensive monkey eye topically with one of these compounds as would be done for glaucoma therapy, causes a substantial reduction in IOP () (Okka et al., 2004
; Peterson et al., 2000
). The outflow facility effect following topical treatment does not start right away but only develops with continued perfusion even though the drug concentration in the anterior chamber is decreasing with time (Okka et al., 2004
; Peterson et al., 1999
). Thus the system has been weakened such that the pressure gradient and fluid flow across the system can now collapse the “house of cards” that the drug initially created. This is a potentially wonderful situation for glaucoma therapy where an even greater pressure gradient is present.
Figure 5 Effects of Latrunculin (LAT) B on IOP and outflow facility in monkeys. A,B. 0.005/0.01% Lat-B and vehicle (4×5μl or 2×10μl) were administered to opposite eyes topically twice daily for 4.5 days. IOP was measured before (more ...)
On the kinase side, H-7, which is available off the shelf, is a rather nonselective Rho kinase/myosin light chain kinase inhibitor, and a protein kinase C inhibitor (Citi et al., 1994
). Y-27632 is a more selectively a Rho kinase inhibitor. Both reduce actomyosin-driven contractility resulting in the deterioration of actin microfilament bundles, perturbation of membrane anchorage of the microfilament system and loosening or weakening of cell-extracellular matrix junctions. Intracameral exchange with either of these compounds results in a dramatic several fold increase in outflow facility in the nonhuman primate eye after an initial delay. Similarly topical H-7 produced about the same doubling of outflow facility (Tian et al., 1998
; Tian et al., 2004
To examine the structural effects, we are working with Dr. Geiger’s group. Following intracameral exchange of monkey eyes with latrunculin B (0.5μM) there is expansion of the juxtacanalicular area, separation of inner wall endothelium along with the first subendothelial cell layer such that it lifts away from the juxtacanalicular meshwork resulting in a ballooning of the juxtacanalicular region (). The entire meshwork is expanded allowing fluid to get through more easily as shown with tracer studies. However there are no breaks in the inner wall itself (Sabanay et al., 2006
Figure 6 Morphology after perfusion of monkey eyes in vivo with H-7 or LAT-B. Light micrographs of vehicle (A) and H7 (B) treated eyes showing expanded intercellular spaces (arrow), extended IW cells, and maintained cell-cell junctions after H-7. (bar = 50μm) (more ...)
H-7 treatment also produces dilation of Schlemm’s canal and expansion of the juxtacanalicular meshwork as the endothelial cells are relaxed, as shown in , which is a schematic 15 cell stretch of Schlemm’s canal inner wall endothelium where the cells are more relaxed and much longer after H-7 treatment (Sabanay et al., 2000
). Tracer injected into the anterior chamber is spread evenly along the inner wall as opposed to the untreated eye (Sabanay et al., 2000
) in which there is a funneling of fluid flow through preferential channels (Johnson et al., 1992
; Overby et al., 2002
), again without inner wall breaks. Thus with these cytoskeletal compounds, the entire wall becomes available for filtration.
The effects of H-7 (Sabanay et al., 2004
) and Lat-A (Peterson et al., 1999
) are reversible, indicating they are due to alterations in cellular contractility and cytoskeletal organization rather than irreversible toxicity. In addition, intravitreal administration of H-7 or latrunculin-B at doses that increase outflow facility and lower IOP when given intracamerally, had no effect on retinal vascular permeability, retinal electrophysiology, or the clinical or angiographic appearance of the retina (Kiland et al., 2006
Interestingly the cornea is not affected at concentrations of these compounds that affect the TM and increase outflow facility (Okka et al., 2004
; Sabanay et al., 2006
). Perhaps there is a differential sensitivity to different cell types. Or, since the corneal endothelium sits on a hard backing, Descemet’s membrane, whereas the TM is basically suspended like a hammock between two fluid compartments at different pressures, the corneal endothelium is not comparably exposed to the concomitant assault by pressure and shear stress.
Derivatives of all these compounds are now being tested in clinical trials. For the latrunculins, it is the first time such molecules have ever been in man. Thus the science has evolved from hard-core cell biology, through organ culture and in vivo animal physiology, and into the clinical arena, involving multinational collaborations and friendships along the way.