|Molecular Vision 2005;
Received 9 March 2005 | Accepted 26 April 2005 | Published 27 April 2005
Expression of dominant negative Rho-binding domain of Rho-kinase in organ cultured human eye anterior segments increases aqueous humor outflow
Ponugoti Vasantha Rao,1,2
PeiFeng Deng,1 Rupalatha Maddala,1 David L. Epstein,1
Chuan-Yuan Li,3 Hiroaki Shimokawa4
Departments of 1Ophthalmology, 2Pharmacology and Cancer Biology, and 3Radiation Oncology, Duke University School of Medicine, Durham, NC; 4Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Science, Fukuoka, Japan
Correspondence to: P. Vasantha Rao, PhD, Department of Ophthalmology, Duke University Medical Center, Box 3802, Durham, NC, 27710; Phone: (919) 681-3237; FAX: (919) 684-8983; email: email@example.com
Purpose: Based on pharmacological inhibition of activity, a role has been proposed for Rho-kinase in the modulation of aqueous outflow and intraocular pressure (IOP). This study employed a molecular approach to specifically address the role of Rho-kinase in the modulation of aqueous humor outflow.
Methods: Adenoviral vectors expressing green fluorescent protein alone (Ad-GFP) or the dominant negative Rho-binding domain of Rho-kinase and GFP (Ad-DNRK-GFP) were utilized in these experiments. Human and porcine primary trabecular meshwork (TM) cells were infected with 30 MOI (multiplicity of infection) of Ad-GFP alone or with Ad-DNRK-GFP. Changes in cell shape, actomyosin cytoskeletal integrity, and the status of myosin light chain (MLC) phosphorylation were evaluated. The aqueous outflow facility was determined in organ cultured anterior segments of human cadaver eyes infected with 107 pfu adenoviral vectors (Ad-GFP or Ad-DNRK-GFP) using a constant flow perfusion system.
Results: Expression of DNRK resulted in changes in cell shape (cell rounding, cell-cell detachment) and decreased actin stress fiber and focal adhesion staining in TM cells. These cellular changes were associated with substantially reduced myosin light chain phosphorylation. Additionally, organ cultured human eye anterior segments infected with Ad-DNRK-GFP exhibited a significant increase in the outflow facility (80%, n=9) compared to control anterior segments infected with Ad-GFP (5%).
Conclusions: This study demonstrated that specific inhibition of Rho-kinase activity in trabecular meshwork cells led to alterations in cell shape and presumed contractile properties, and we hypothesize that these morphological and contractile events underlie the observed effects of dominant negative Rho-kinase on the aqueous humor outflow facility. These data provide molecular evidence for the hypothesis of Rho-kinase being a potential cellular target involved in the regulation of aqueous humor outflow resistance, with implications for novel glaucoma therapy.
Glaucoma, a leading cause of blindness resulting from optic nerve damage and atrophy, is commonly associated with increased intraocular pressure (IOP). In most forms of glaucoma, increased intraocular pressure is thought to result from a defective drainage of aqueous humor through the trabecular meshwork and Schlemm's canal [1-3]. A variety of pharmacological compounds have been identified which influence the aqueous humor outflow facility and IOP in both in vitro and in vivo models, however, very little is known about the regulation of aqueous humor outflow through the trabecular meshwork outflow pathway [1-15].
Over the last several years, pharmacological compounds that are known to influence cell shape, cell-cell junctions, and cell-extracellular matrix interactions within the outflow pathway have been recognized to influence the aqueous humor outflow resistance in perfusion studies [4-6,8,10,16]. Additionally, using selective inhibitors of Rho-kinase [12-15], protein kinase C [9,17], and certain physiological agonists of G-protein coupled receptors [11,14], it has been demonstrated that the phosphorylation status of the myosin light chain (MLC) is an important biochemical determinant of aqueous humor outflow facility through the trabecular meshwork outflow pathway. The phosphorylation status of MLC is known to alter cellular contraction and relaxation, cell shape, cell-extracellular matrix (ECM) interactions, and intercellular junctions by regulating actomyosin interactions in endothelial and other cell types [11,12,18-25].
The trabecular meshwork, which is believed to play a major role in conventional aqueous outflow, is reported to have smooth muscle-like properties [2,3,6,14]. The contractile properties of this tissue influence the aqueous outflow facility, perhaps by influencing the size of the filtering spaces in trabecular meshwork (TM), permeability properties of the inner wall of Schlemm's canal [2,6,10,22], and/or via regulation of ECM turnover.
Rho GTPase, the ubiquitously expressed small GTP-binding protein, plays a critical role in various cellular processes, including smooth muscle contraction, actin cytoskeletal organization, cell adhesion, and migration [18,23-25]. Both the organization of the actin cytoskeleton and its interaction with cell adhesion proteins are primarily regulated by Rho GTPase and its downstream effector, Rho-kinase [18,23,24]. Additionally, maintenance of cellular morphology depends on actomyosin cytoskeletal integrity and inactivation of Rho GTPase or Rho-kinase has been shown to induce changes in cell shape, cell-cell, and cell-ECM interactions in various cell types [12,18,23-25]. Interestingly, Rho kinase inhibitor induced increases in aqueous outflow facility has been shown to correlate well with changes in cell shape, decreased MLC phosphorylation, decreased focal adhesions, and actin depolymerization in trabecular meshwork cells [12-14].
However, studies based on either in vivo and in vitro perfusion or on topical eye drug application typically use higher concentrations of pharmacological agents to potentially modulate the activity of target proteins, primarily because of lack of information on the tissue concentration of any given agent and especially because information on their accessibility to the site of the action are not known [5-8,12-15]. Therefore, it is both necessary and important to validate the target specificity of pharmacological agents by independent means. In the case of Rho-kinase, in particular, it is critical to confirm the specificity of the potential role of this signaling protein in regulation of the aqueous outflow facility because of its immense potential as a drug target for lowering intraocular pressure [12-15,26,27]. Furthermore, it is quite possible that aberrant regulation of the Rho GTPase/Rho-kinase pathway could underlie the pathophysiology of glaucoma [11-14].
To determine the specific role of Rho-kinase in modulating aqueous humor outflow through the trabecular meshwork, we applied a molecular approach and expressed a dominant negative Rho-kinase (DNRK) in TM cells in a cell culture model and in the aqueous outflow pathway of organ cultured human eye anterior segments. Using a well characterized adenoviral vector expressing the dominant negative Rho-binding domain of Rho-kinase (DNRK) and green fluorescent protein (GFP) [28,29], we demonstrated the specific effects of dominant negative Rho kinase on the aqueous humor outflow facility in organ cultured human eye anterior segments, and correlated these findings with the status of MLC phosphorylation, cell shape, and actin cytoskeletal organization in human trabecular meshwork cells in vitro.
Trabecular meshwork cell cultures
Human and porcine primary trabecular meshwork cells were isolated from cadaver eyes by collagenase digestion as described previously . Trabecular meshwork cells were cultured at 37 °C and under 5% CO2 using Dulbecco's modified Eagle medium (DMEM) containing 10% bovine fetal serum (FBS), penicillin (100 Units/ml), and streptomycin (100 μg/ml). Cells passaged three to five times and obtained from different donors were used in this study. Based on availability, either porcine or human TM cells were used in this study.
Well characterized replication defective recombinant adenoviral vectors encoding either enhanced green fluorescent protein (GFP) alone or GFP and the mutant Rho-binding (NK1036->TT) and pleckstrin homology (PH) domains of Rho-kinase (Figure 1) were provided by Dr. Shimokawa. Expression of both control GFP and mutant Rho kinase is driven by the cytomegalovirus promoter. Both of these vectors contain a CMV-MCS-IRES-EGFP-poly A insert released from the pIRES (internal ribosome entry site)-EGFP plasmid (Clontech, Palo Alto, Ca). These viral vectors were amplified using 293 cells and purified using the BD Adeno-XTM Virus purification kit (BD Bioscience, Palo Alto, CA). The titer of the virus stock was assessed by a plaque formation assay using 293 cells and expressed as plaque forming units (pfu).
Primary human or porcine TM cells cultured to 70% confluence in plastic cell culture plates were infected with adenoviral vectors expressing either GFP alone or DNRK and GFP at 30 MOI in the absence of serum. Two hours after the addition of adenoviral vectors, the culture medium was supplemented with serum to obtain a final concentration of 10% serum. Expression of GFP was evaluated by recording the green fluorescence using a phase contrast microscope with a fluorescent light source.
TM cells grown on gelatin coated glass coverslips were infected with adenoviral vectors expressing either GFP alone or GFP plus DNRK at 30 MOI. Changes in cell shape were recorded using phase contrast microscopy (Zeiss IM 35), and after an adequate portion (up to 80% of total number of cells) of the total number of cells exhibited expression of GFP (about 48 h after infection), cells were fixed for immunofluoresence analysis of actin cytoskeletal organization and focal adhesion formation, as described previously [11,12]. Actin and focal adhesions were stained with rhodamine phalloidin or vinculin primary antibody, in conjunction with rhodamine conjugated secondary antibody, respectively. Representative fluorescence (for actin) and immunofluoresence (for vinculin) images were recorded using Zeiss Axioplan2 fluorescence microscopy. These analyses were carried out in two independent experiments and in multiples.
Myosin light chain phosphorylation
The status of myosin light chain (MLC) phosphorylation in human TM cells infected with adenoviral vectors (30 MOI for 48 h) was determined by western blot analysis, as described previously [11,12], using a phosphospecific MLC (Thr18/Ser19) polyclonal antibody obtained from Cell Signaling Technology, Inc. (Beverly, MA). In addition to analyzing equal amounts of total protein from different samples, we also analyzed levels of the housekeeping protein actin, to normalize for protein loading in MLC phosphorylation assays. Densitometric analysis was performed to quantify the DNRK induced changes in MLC phosphorylation, using NIH Scion Image software. Data based on three independent experiments were tested for statistical difference using the Student t-test.
Organ culture perfusion
Cadaver human eyes were obtained from NDRI (National Disease Research Interchange, Philadelphia, PA) or the North Carolina Eye Bank. Eyes were obtained within 48 h of death from donors aged between 38 and 80 years, and none of the eyes used in this study were diagnosed as glaucomatous. The use of human tissue in this study was in accordance with the guidelines of the Declaration of Helsinki, and the only donor details provided were age and sex of the donor. Anterior segments were prepared by dissecting the eyes at the equator. The lens, iris, and vitreous were removed and rinsed thoroughly with culture media before clamping the anterior segments to a two cannulae, modified Petri dish, as described by Vittitow et al.  Anterior segments were perfused with DMEM media containing 0.1% FBS, penicillin (100 units/ml), streptomycin (100 μg/ml), gentamicin (36 μg/ml), and amphotericin (0.25 μg/ml) at a constant flow (3 μl/minute) and under 5% CO2 at 37 °C, using Harvard microinfusion pumps (Harvard Bioscience, South Natick, MA). Intraocular pressure was monitored continuously with a pressure transducer connected to the dish's second cannulae and recorded with an automated computerized system.
Anterior segments were initially perfused with culture medium for 24 h during which the initial baseline pressure was monitored on a continuous basis. After this, perfusion pumps were stopped briefly to lower the pressure in the anterior chamber of the eye, and adenoviral vectors expressing either GFP alone, or GFP and DNRK, were injected intracamerally in 20 μl volume containing 107 pfu. Following viral vector injections, Petri dishes were gently swirled to disperse the viral vectors and perfusion was continued at a constant flow (3 μl/minute) for six days. Effects of DNRK on outflow facility were expressed as the percentage change in outflow facility (compared to baseline values) over 72 h. Values were expressed as mean±SEM, and a paired two tailed Student's t-test was applied to determine the significance of difference in outflow facility between control and DNRK expressed. Eyes that did not maintain a stable baseline intraocular pressure during the initial 24 h perfusion with medium alone, were excluded from the perfusion study.
GFP expression in organ cultured anterior segments and in aqueous outflow pathway
At the end of perfusion, anterior segments, expressing the GFP alone or GFP plus DNRK, were removed from the perfusion system and placed in Petri dishes with the cornea facing down and the ciliary body facing up. Expression of GFP in the perfused eye was evaluated by phase contrast microscopy. Images of distribution of green fluorescence in different regions of the anterior segment, including cornea, aqueous outflow angle, and ciliary body were recorded with a camera attached to the microscope.
To determine the distribution and expression of adenoviral vector driven DNRK and GFP in aqueous outflow pathway, organ cultured human eye anterior segments infected with adenoviral vectors expressing either DNRK and GFP or GFP alone were dissected into 4 quadrants at the end of the six day perfusion period. Tissue sections were initially fixed in 4% paraformaldehyde prepared in phosphate buffered saline (PBS) for 24 h and then stored in 5% sucrose in PBS and 30% sucrose in PBS for 24 h sequentially at 4 °C. To obtain cryosections, the tissue was embedded in Tissue-Tek Optimum Cutting Temperature compound (Sakura, Torrance, CA) at -20 °C and sectioned (5-10 μm thickness) using a cryomicrotome (Microm HM 550). Tissue sections were placed on glass slides and mounted with fluoromount-G and viewed under the fluorescence microscope. Images were captured using a Zeiss fluorescence microscope.
At the end of six days of perfusion following infection with viral vectors, organ cultured anterior segments were unclamped from the perfusion chambers and the tissue was dissected into 4 quadrants under a dissecting microscope. Tissue was then fixed with 2.5% glutaraldehyde and 2% formaldehyde at room temperature as described previously , for evaluating histological changes in the outflow pathway. Tissue quadrants obtained from control (GFP alone) and DNRK expressing eyes were fixed in 1.0% osmium tetra oxide in 0.1 M sodium cacodylate buffer and then stained with 1% uranyl acetate. Finally, microtome sections were stained with KMnO4 and Sato's stain, and photographed using both light and electron microscopes (Joel Jem-1200 EX).
Effects of DNRK expression on TM cell morphology and cytoskeletal organization
Human and porcine TM cells infected with adenoviral vectors expressing GFP plus DNRK demonstrated changes in cell shape (cell rounding and cell-cell separation). Expression of GFP was noted within 24 h, and 80% of cells were found to express GFP within 48 h after exposure to viral vectors in both control cells (treated with vectors expressing GFP alone) and test cultures (treated with vectors expressing GFP and DNRK). Unlike control cells, those treated with DNRK plus GFP exhibited marked changes in cell shape, including cell-cell detachment, cell rounding, and formation of a filamentous cell body Figure 2A,B. DNRK treated cells exhibited obvious loss of actin stress fibers and reduction in vinculin staining when compared to GFP expressing control cells, indicating alterations in actin cytoskeletal organization and focal adhesions (Figure 2B). Control cells expressing GFP alone exhibited normal cell morphology and actomyosin organization was indistinguishable from untreated control cells (Figure 2A). These analyses were carried out using confluent cell cultures expressing either GFP alone or DNRK and GFP.
TM cells expressing GFP or DNRK and GFP for 24 to 72 h after viral vector infection did not exhibit any cell death or damage as assessed by propidium iodide incorporation. However, at 92 h, a small percentage of cells exhibited positive staining in the nucleus for propidium iodide suggesting some cell death or cell damage (data not shown).
Effects of DNRK expression on MLC phosphorylation in TM cells
Myosin phosphatase, which regulates MLC phosphorylation, is a well studied target of Rho-kinase. Phosphorylation of myosin phosphatase by Rho-kinase leads to inactivation of its phosphatase activity, and causes an increase in MLC phosphorylation which then produces cellular contraction [20,25,31,32]. To determine the effects of expression of DNRK on MLC phosphorylation in TM cells, cell lysates obtained from human TM cells infected with GFP alone or DNRK plus GFP were analyzed for changes in MLC phosphorylation by western blot analysis using a phosphospecific MLC antibody (reacts specifically with the diphospho-form of MLC). TM cells expressing DNRK exhibited a marked reduction in MLC phosphorylation compared to GFP expressing control cells or untreated control cells, based on analysis of equal amounts of total protein (Figure 3A). Densitometric analysis based on three independent experiments revealed a significant decrease (65%, p<0.0003) in MLC phosphorylation in DNRK expressing cells compared to cells expressing GFP alone (Figure 3B), and a 45% decrease (p<0.05) compared to untreated cells. TM cells expressing the GFP control showed slightly higher levels of MLC phosphorylation when compared to untreated cells (Figure 3B). The difference in MLC phosphosphorylation between untreated and GFP treated TM was not significant. In addition to using equal amounts of total protein from control and DNRK expressing samples, actin levels were also determined by western blot analysis to confirm equality of protein loading (Figure 3A, lower panel).
Changes in aqueous outflow facility induced by DNRK expression
Expression of DNRK in organ cultured anterior segments of human eyes led to a significant increase in the aqueous outflow facility compared to control eyes expressing GFP alone. Figure 4 depicts the percent change in the aqueous humor outflow facility from baseline values in control and DNRK expressing samples, with the change in facility between control and DNRK expressing eyes being statistically significant (p<0.006) from 12 h after perfusion to 72 h. The mean baseline outflow facility (μl/min/mm Hg) in control and DNRK expressing eyes was 0.253±0.0263 and 0.158±0.0258 (n=9, values are mean±SEM), respectively. After 72 h of viral infection, the mean outflow facility in control and DNRK expressing eyes was 0.254±0.0345 and 0.267±0.0359, respectively. The percent change in outflow facility from baseline in DNRK expressed eyes was increased by 80% at 54 h after perfusion (p<0.002), and this response was found to be progressive until 72 h (p<0.006). The aqueous humor outflow facility was increased by 60% at six days after perfusion (data not shown). On the other hand, in control eyes expressing only GFP, the percent change in facility from baseline values changed only marginally and was found to be around a 5% increase from the baseline at 48 h after perfusion. Out of nine individual anterior segments infected with DNRK, 8 samples demonstrated a progressive increase in the outflow facility during the perfusion period, whereas one sample showed an inconsistent increase in the outflow facility.
Expression of DNRK in organ cultured anterior segments
To detect the expression pattern of DNRK in organ cultured anterior segments of human eyes, adenoviral vector infected anterior segments (used in determining the changes in outflow facility) were examined for the expression of GFP after completion of perfusion. GFP expression was evaluated by microscope aided analysis of the distribution pattern of green fluorescence in tissues of the anterior segments. Figure 5A illustrates the expression and distribution pattern of GFP in different tissues of the anterior segment of the human eye. In both control and DNRK infected anterior chambers, GFP expression was observed in corneal endothelium and ciliary body. However, when compared to these tissues, there was an intense GFP fluorescence observed in the aqueous outflow angle in both control and DNRK treated eyes (indicated with arrows in Figure 5A). Additionally, analysis of 7 μm cryosections derived from these same samples revealed an intense GFP fluorescence in TM tissue from both control and DNRK samples (Figure 5B), confirming adenoviral driven expression of DNRK in the cells of the aqueous outflow pathway in organ cultured eyes.
Integrity of the aqueous outflow pathway in DNRK expressing human eye anterior segments
To evaluate integrity of the aqueous outflow pathway including TM and SC in GFP and DNRK-GFP expressing eyes, we performed both light and transmission electron microscope based histological analysis using 2 test (DNRK expressing) and 2 control (GFP expressing) eyes. The 2 DNRK samples exhibited a definite increase in the aqueous outflow facility. Light microscopic analysis of different quadrants derived from DNRK-GFP or GFP expressing specimens revealed comparable histological integrity in the region of TM and SC (Figure 6A,D), with no marked difference between the two groups. Transmission electron microscopic analysis, however, revealed some discontinuity of inner and outer wall lining of SC in both control (GFP alone) and test (DNRK and GFP) specimens as depicted by the black arrow heads in Figure 6B,C,E,F. Additionally, detachment of SC cells from the subendothelial region of juxtacanicular tissue (JCT; labeled with black stars) was noted in both groups of eyes. There seemed to be prominent breaks in the inner wall of SC in the test group as compared to the control group. TM cell morphology in the outer TM region appears to display subtle changes (cells appear round), and TM cells were detached from the TM lamellae in DNRK expressing specimens (indicated with white arrow heads) compared to GFP expressing specimens (Figure 6D,E).
Rho-kinase, a well characterized downstream effector of the small GTPase, Rho, plays a critical role in the regulation of contractile properties of smooth muscle cells in a calcium independent fashion, via modulation of the MLC phosphorylation status [25,31-33]. In this study, we demonstrated the specific effect of Rho-kinase inhibition in increasing the aqueous humor outflow facility using a molecular approach wherein adenoviral vector mediated overexpression of a dominant negative mutant form of Rho-kinase was employed to inhibit endogenous Rho-kinase function. Expression of DNRK in TM cells led to dramatic changes in TM cell shape, decreased formation of actin stress fibers and focal adhesions, and inhibition of MLC phosphorylation. These cellular and biochemical changes were correlated with detectable increases in the aqueous humor outflow facility in organ cultured human anterior segments in DNRK expressing specimens.
Rho-kinase, an important downstream effector of activated RhoA in smooth muscle, is one of the critical regulators of myosin light chain phosphatase (MLCP), which directly influences the status of MLC phosphorylation [18,23-25,31-33]. It is well documented that Rho and its downstream target, Rho-kinase, play an important role in Ca2+ independent regulation of vascular smooth muscle contraction, actomyosin organization, and cell-ECM interactions [25,31-33]. Furthermore, the Rho/Rho-kinase pathway is also recognized to play an essential role in several physiological processes, and the aberrant activity of this signaling pathway has been associated with various pathological conditions such as hypertension and vasospasm [33-35]. These findings have generated a strong interest in Rho-kinase as a target for disease therapy [33-35].
In our previous studies based on the use of selective pharmacological inhibitors, we have reported a potential role for Rho-kinase and Protein kinase C in the regulation of MLC phosphorylation and the aqueous outflow facility [11,12,14,17]. Using various physiological agonists of G-protein coupled receptors, including endothelin-1, thromboxane A2, Angiotensin II, and lysophospholipids, we have documented the link between ligand mediated activation of Rho GTPase, increased MLC phosphorylation in TM cells, and decreased aqueous outflow facility in perfused porcine eyes [11,14]. Based on these and other studies, it became apparent that the Rho/Rho-kinase pathway might play a critical role in the regulation of aqueous outflow under physiological conditions and that Rho-kinase could serve as a potential target for therapeutic approaches designed to lower the intraocular pressure in glaucoma patients [12-15,26,27,36].
To explore such a possibility and to address a specific role for Rho-kinase in the modulation of the aqueous outflow facility, we sought to obtain independent confirmation of our earlier findings which were based on pharmacological studies [12,14]. This was accomplished using a well characterized adenoviral vector encoding a dominant negative Rho-kinase in conjunction with the GFP [28,29] to achieve overexpression of DNRK in TM cells and intact anterior segments derived from human samples. We then evaluated in cell culture the effects of overexpressing DNRK on TM cell shape, actomyosin cytoskeletal organization, and MLC phosphorylation and compared these results to those in control TM cells expressing GFP alone. Replication-defective recombinant adenoviral vectors expressing the dominant negative Rho-binding domain of Rho-kinase and enhanced GFP (Ad-DNRK-GFP) or GFP alone (Ad-GFP) were utilized to infect either TM cells or anterior segments from human samples, and the expression pattern of DNRK was assessed based on GFP fluorescence. As shown in Figure 2, TM cells infected with either Ad-DNRK-GFP or Ad-GFP alone showed intense GFP fluorescence within 24 to 36 h after infection. Compared to control cells expressing GFP alone, cells expressing DNRK and GFP demonstrated a dramatic change in cell shape and actin cytoskeletal organization (Figure 2B), effects similar to those induced pharmacologically by Rho-kinase inhibitors [12,14]. Trabecular meshwork cells expressing DNRK also showed a significant decrease in MLC phosphorylation, as shown by western blot analysis using a phosphospecific anti-MLC antibody (Figure 3).
MLC phosphorylation status is an important determinant of cellular contractile status. Increases or decreases in MLC phosphorylation mediate enhancement of cellular contraction and relaxation properties, respectively [20,21,25]. Therefore, the DNRK associated decrease in MLC phosphorylation in TM cells is suggestive of induced cellular relaxation, an effect which is consistent with the observed changes in actin cytoskeletal organization and focal adhesions in these cells (Figure 2B).
Adenoviral vectors injected into organ cultured human eye anterior segments showed intense GFP expression in the anterior chamber angle and TM tissue, in both Ad-DNRK-GFP and Ad-GFP treated eyes (Figure 5A,B). In addition to the anterior chamber angle, the ciliary body and cornea also showed some expression of GFP in both control and DNRK samples. Anterior segments infected with Ad-DNRK-GFP exhibited a significant increase in the aqueous outflow facility at 12 h after injection, compared to control anterior segments expressing Ad-GFP alone. The aqueous facility was increased by up to 80% over baseline values by 50 h after injection with Ad-DNRK-GFP. In contrast, the outflow facility was altered only marginally (<5%) in control eyes infected with Ad-GFP alone. A previous study by Vittitow el al.  reported an increase in the aqueous outflow facility in organ cultured human eye anterior segments overexpressing dominant negative Rho GTPase, the upstream regulator of Rho-kinase. However, the percent change in the facility found in this study (80%) with expression of DNRK was much greater than that reported by Vittitow et al.  with dominant negative Rho GTPase (30%). This difference between outflow facility effects of inhibiting Rho compared to Rho-kinase function indicates that direct targeting of Rho-kinase represents a more effective strategy for potentially modulating aqueous outflow. Both of these observations underscore the potential importance of the Rho/Rho-kinase pathway in the regulation of aqueous humor outflow through the TM, and demonstrate that selective inactivation of Rho-kinase leads to increased outflow (Figure 4).
It is notable that the morphology of the aqueous outflow pathway including the TM, JCT, and inner wall of Schlemm's canal showed only subtle differences between control (infected with Ad-GFP alone) and experimental (infected with Ad-DNRK-GFP) specimens in this study (Figure 6). Interestingly, even though we found discontinuities of the lining of the inner wall of SC in both experimental and control anterior chambers, the breaks in the inner wall of SC in DNRK expressing samples appear to be much more prominent as compared to those in the GFP expressing specimen. This subtle difference in SC ultrastructure might not, however, explain the huge increase in outflow facility associated with DNRK expression. Additionally, a recent study by Bahler et al.  reported that the discontinuous inner wall lining of SC or breaks in this region caused by pharmacological agents do not correlate well with expected alterations in the aqueous outflow facility. Both in cell cultures and in perfusion models, the expression of DNRK influences TM cell morphology (Figure 2 and Figure 6D-F), and in organ cultured eyes (Figure 6D-F) we also found some detachment of TM cells from the TM beams. Whether these changes are involved in DNRK induced increases in outflow facility is a subject of speculation at present since we did not undertake quantitative analyses of the data generated. Therefore, additional studies involving the live animal model might provide further insight into the role of Rho kinase activity in the regulation of aqueous humor outflow.
In conclusion, the data from this study demonstrate that inhibition of Rho-kinase can very specifically increase the aqueous humor outflow facility through the trabecular meshwork outflow pathway, most likely by decreasing MLC phosphorylation and thus altering cytoskeletal integrity, events which in turn regulate cellular contractile and relaxation properties. Furthermore, these data provide molecular evidence for the hypothesis of Rho-kinase being a potential cellular target involved in the regulation of aqueous humor outflow resistance, with implications for novel glaucoma therapy.
Presented in part at the Annual Meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, FL, April 2004. This work was supported by NIH/NEI grants EY013573, EY12201 (PVR), P-30 EY05722, and funding from Research to Prevent Blindness.
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