Limbal fibroblast conditioned media: a non-invasive treatment for limbal stem cell deficiency.

PURPOSE: Limbal fibroblasts are known to regulate the maintenance and differentiation of the corneal epithelium including the limbal epithelial stem cells. This study examined the effect of limbal fibroblast conditioned media in a mouse model of limbal stem cell deficiency. METHODS: Limbal stem cell deficiency was created in C57/Bl6 mice by performing a limbus to limbus epithelial debridement. The mice were treated topically for 3 weeks with conditioned media derived from human limbal fibroblasts. The control mice were treated with skin fibroblast conditioned media or Dulbecco's serum-free medium. RESULTS: The mice treated with limbal fibroblast conditioned media demonstrated substantial growth of corneal type epithelial cells on the corneal surface with less conjunctival goblet cells. By contrast, the control treated corneas were found to be covered primarily by conjunctival type epithelium. CONCLUSIONS: Cell culture media conditioned by limbal fibroblasts appear to contain factor(s) that are therapeutically beneficial in a model of limbal stem cell deficiency. The current results further support the notion that the essential limbal stem cell niche is provided by limbal fibroblasts and suggest a new, non-invasive option in the treatment of limbal stem cell deficiency.

Down-regulation of Notch signaling during corneal epithelial proliferation.

PURPOSE: We evaluated the expression and activation of Notch pathway genes in the adult human and murine corneal epithelium during proliferation. METHODS: The expression of Notch pathway genes in the limbal and central human corneal epithelium was compared by reverse transcription polymerase chain reaction (RT-PCR). Their expression pattern was examined by immunofluorescence and in situ hybridization. The temporal expression of Notch1 during murine wound healing was assessed by RT-PCR. Notch activity was determined using western blot for the Notch intracellular domain (NotchIC). The expression of Hes1 was evaluated in cell culture. RESULTS: The expression of Notch1 and Jagged1 was higher in the human limbal epithelium while the expression of Hes1 and Hes5 was higher in the central cornea. Expression of Notch1, Jagged1, and Hes1 was found predominantly in the basal and immediate suprabasal cells. During neonatal corneal development, NotchIC was detected in occasional cells at P10 while at P15 and P90, it was found in the basal and early suprabasal layers. NotchIC was found to be lower in the limbal compared to central corneal epithelium. The expression of Notch1 was lower at 24 h post-wounding but was completely restored in six days. The levels of NotchIC were decreased at 24 h post-wounding and after application of topical phorbol myristate. In vitro, the expression of Hes1 was higher in confluent cells maintained under high calcium conditions. CONCLUSIONS: The inverse correlation between Notch signaling and the proliferative status of the corneal epithelium is consistent with the idea that Notch plays a role in corneal epithelial differentiation.

The synthesis of glycosaminoglycans by cultures of corneal stromal cells from patients with keratoconus.

Keratoconus is a disease that results in thinning and ectasia of the central cornea. Cultures of corneal stromal cells from patients with keratoconus were established and the synthesis of glycosaminoglycans compared with the synthesis of glycosaminoglycans by normal human corneal stromal cells in culture. Keratoconus and normal control cell cultures were incubated with sodium [(35)S]sulfate and [(3)H]glucosamine for 4 h. After incubation, the labeled glycosaminoglycans were isolated from the medium fractions and cells. Keratoconus and normal control cultures synthesized similar amounts of sulfated glycosaminoglycans independent of the age of donors and(or) the number of subcultures. In contrast to normal control cultures, most of the newly synthesized glycosaminoglycans produced by keratoconus cells were found in the growth medium and much less were in the cell layer. Treatment with glycosaminoglycan-degrading enzymes followed by paper chromatography showed that keratoconus cells, as normal control cells, produced hyaluronic acid and various sulfated glycosaminoglycans. The production of cell layer-related heparan sulfate was markedly reduced in keratoconus cultures. Because heparan sulfate has been shown to be associated with cell surfaces, the decreased heparan sulfate content could reflect changes at this location.

Identification of collagens synthesized by cultures of normal human corneal and keratoconus stromal cells.

We have examined the collagens synthesized by cultures of normal human corneal stromal cells. Radioactively labeled products, accumulated in the culture medium during a 24-h labeling period, were treated with pepsin and analyzed by SDS-polyacrylamide gel electrophoresis. The cell layer collagen was characterized by 2.6 M and 4.4 M salt fractionation at neutral pH. CM-cellulose column chromatography, SDS-gel electrophoresis, and cyanogen bromide peptide mapping. Type I alpha 1 and alpha 2 chains were the predominant components in both the cell layer and the medium fractions of normal human stromal cultures; type III collagen was found mostly in the culture medium; and type V collagen was associated with the cell layer. Immunofluorescent techniques used to visualize collagen deposition in the cell layer confirmed the presence of these collagen types. Keratoconus is a disease characterized by thinning and scarring of the central cornea. Stromal cells grown from keratoconus corneas produced similar types of collagen (types I, III, and V) as normal human controls. Cells from keratoconus patients, however, contained more type V collagen in the cell layer than did normal cells. The difference was seen only in the 4.4 M salt precipitates. Since type V collagen is one component of cell surfaces, the primary defect in cultures from keratoconus corneas could involve cell membrane and cell surface components.

Histopathological and immunohistochemical studies of lenticules after epikeratoplasty for keratoconus.

AIMS: To examine histopathological and immunohistochemical changes in lenticules and host of corneal buttons from patients who previously underwent epikeratoplasty for keratoconus. METHODS: 12 penetrating keratoplasty specimens from patients with keratoconus who had previously undergone epikeratoplasty, eight keratoconus, and seven normal corneas were examined. Immunostaining for Sp1, alpha1-proteinase inhibitor (alpha1-PI), and alpha2-macroglobulin (alpha2M) were performed. RESULTS: In nine of the 12 lenticules, the keratoconus-like disruptions were found in Bowman's layer. Peripheral and posterior keratocyte repopulation of the lenticules was observed in all cases. Keratocyte repopulation in the anterior and mid-stromal regions of the lenticules appeared related to the time since epikeratoplasty. Sp1 nuclear staining of the basal and wing epithelial cells was more intense in lenticules and keratoconus corneas than in normal corneas. Lenticular, host, and keratoconus keratocytes showed positive Sp1 staining, whereas staining was absent in normal corneas. Compared to normal corneas, alpha1-PI and alpha2M immunostaining was lower in the lenticules, host, and keratoconus specimens. CONCLUSIONS: The epithelial cells and keratocytes repopulated in the lenticules retain keratoconus-like biochemical abnormalities such as upregulation of Sp1 and downregulation of alpha1-PI and alpha2M. The authors speculate that both keratocytes and the corneal epithelium may participate in the development of keratoconus.

Ultrastructural localization of myocilin in human trabecular meshwork cells and tissues.

We examined ultrastructurally the localization of myocilin (formerly called trabecular meshwork inducible glucocorticoid response, or TIGR) protein in cultured human trabecular meshwork (TM) cells and in normal human TM tissues. The TM, a specialized tissue located at the chamber angle of the eye, is believed to be responsible for the development of glaucoma. The myocilin gene has been directly linked to both juvenile and primary open-angle glaucomas, and multiple mutations have been identified. Human TM cells were treated with 0.1 mM of dexamethasone (DEX) to induce myocilin expression. This protein was immunolocalized by colloidal gold electron microscopy using an anti-human myocilin polyclonal antibody. Double labeling with different sizes of gold particles was also performed with additional monoclonal antibodies specific for cell organelles and structures. In both DEX-treated and untreated cultured cells, myocilin was associated with mitochondria, cytoplasmic filaments, and vesicles. In TM tissues, myocilin was localized to mitochondria and cytoplasmic filaments of TM cells, elastic-like fibers in trabecular beams, and extracellular matrices in the juxtacanalicular region. These results indicate that myocilin is localized both intracellularly and extracellularly at multiple sites. This protein may exert diverse biological functions at different sites.

Effects of corneal extracts on rabbit corneal stromal cells in culture.

Rabbit corneal tissues were treated sequentially with phosphate buffered saline (PBS) and 4 M guanidine hydrochloride (GuHCl), dialyzed, and lyophilized. The interaction of the individual PBS and the GuHCl extracts with cultured rabbit corneal stromal cells was assessed. The PBS extract stimulated stromal cell growth. These cells had a thinner spindle-shaped appearance, a greater tendency toward multilayer formation, and a approximately 40-60% higher final density than the controls. The cells subjected to the GuHCl extract exhibited no such changes. When the PBS extract was heated to 80 degrees C, the stimulatory activity was replaced by an inhibitory activity, indicating that the PBS extract contained both the stimulatory and the inhibitory factors. Using a high performance liquid chromatograph system, such factors could be separated. The effects of corneal extracts on connective tissue synthesis were examined after labeling confluent stromal cultures with either (35S)sulfate or (3H)proline for 20 hr. The PBS and the GuHCl extracts significantly promoted the incorporation of (35S)sulfate into glycosaminoglycans. Neither extract altered the types of glycosaminoglycans synthesized or the collagen synthesis of stromal keratocytes in culture.

Adhesion of human trabecular meshwork cells to extracellular matrix proteins. Roles and distribution of integrin receptors.

PURPOSE: To examine the adhesion of human trabecular meshwork cells with various extracellular matrix (ECM) proteins and to evaluate the roles and distribution of integrin receptors. METHODS: Cultured human trabecular meshwork cells were added to 96-well plates either uncoated or coated with proteins including fibronectin, laminin, and vitronectin, as well as collagen types I, III, IV, V, and VI. After incubation for 1 hour, the adhesion of cells was measured. Expression of cell surface integrins was determined by cell enzyme-linked immunoadsorbent assay (ELISA) and immunoprecipitation. Distribution was visualized by immunofluorescence staining using integrin-specific antibodies. For function perturbation and peptide inhibition studies, cells were preincubated with either integrin antibodies or synthetic peptides before the adhesion assays. RESULTS: Human trabecular meshwork cells attached to plates coated with ECM proteins in a dose-dependent manner. Fibronectin, vitronectin, and collagen types I and IV were the preferred ECM substrates. Cell ELISA revealed the presence of integrins alpha 1, alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, alpha v, beta 1, beta 3, beta 5, alpha 5 beta 1, and alpha v beta 3 and the absence of beta 2 and beta 4 on human trabecular meshwork cells. Results from immunostaining experiments were consistent with those from cell ELISA studies. Attachment of cells to ECM proteins was blocked by specific integrin antibodies. Cell adhesion to fibronectin and vitronectin was inhibited by peptides containing the Arg-Gly-Asp sequence. CONCLUSIONS: Extracellular matrix proteins mediate the adhesion of human trabecular meshwork cells in culture. Integrin receptors appear to have functional roles in the cell-matrix interactions.

Effects of ascorbic acid on cultured rabbit corneal endothelial cells.

Rabbit corneal endothelial primary cultures and subcultures with initial high seeding densities (split ratio 1 : 2) and low seeding densities (split ratio 1 : 4) were maintained routinely in Falcon Petri dishes. Ascorbate, 75 microgram/ml, was added daily to experimental cultures to evaluate its effect on growth of endothelial cells. With supplemental ascorbic acid, the cells grew at a slow rate, had a shorter lifespan, and reached a lower cell density at confluency than did control cells. The inhibitory effect of ascorbic acid on cell growth was more pronounced with cultures plated at low cell densities and with cells in later passages. Cultures grown with ascorbate contained a greater number of elongated cells and large-sized cells with vacuoles. A basal lamina was observed even in control cultures with no supplemental ascorbate.

Growth of human corneal endothelial cells in culture.

We investigated the effects of various culture conditions on the growth of normal human corneal endothelial cells in culture. Falcon Primaria tissue culture plastic was found to provide a more suitable surface for endothelial cell growth than the conventional Corning tissue culture plastic. Also, media containing 10% fetal bovine serum and 5% calf serum (complete media) facilitated the growth of human cells better than those containing Nu-serum. Supplementation with epidermal or fibroblast growth factor (10 and 100 ng/ml) to the complete media had no effect on human endothelial cell growth. Chondroitin sulfate at low concentrations (100 micrograms/ml to 1 mg/ml) also showed little effect. At high concentrations (13.5 and 25 mg/ml), however, chondroitin sulfate significantly promoted human corneal endothelial cell growth during a 1- to 2-week incubation period. From the 37 cultures initiated, outgrowth from explants appeared within 3 to 7 days. Cells were polygonal in shape and, at confluency, formed a continuous monolayer. We attained a success rate of 87% (7/8) growing primary cultures from donors under 20 years of age and a 59% (17/29) success rate from older donors.

Sulfated proteoglycans in the human lamina cribrosa.

The sulfated proteoglycans in the normal human lamina cribrosa were studied by electron microscopy after cuprolinic blue dye binding. Within the cores of the laminar plates, three types of cuprolinic blue-positive proteoglycan filaments with different sizes were associated with collagen fibers. These filaments, which were partially sensitive to chondroitinase AC and chondroitinase B, were completely removed by chondroitinase ABC and were identified as chondroitin/dermatan sulfate proteoglycans. In addition, small punctate and filamentous structures that stained with cuprolinic blue were associated with the basal laminae of astrocytes and blood vessels. Enzyme chondroitinase ABC had no effect, but heparinase digested all of these basement membrane-associated structures, indicating that they represented heparan sulfate proteoglycan molecules. Keratanase did not affect any of the cuprolinic blue-positive materials. This investigation illustrates the ultrastructural distribution and morphology of proteoglycans in the human lamina cribrosa and provides baseline information for future studies regarding the roles of proteoglycan molecules in diseases such as glaucoma.

Synthesis of glycosaminoglycans by cultures of normal human corneal endothelial and stromal cells.

Monolayer cultures of normal human corneal endothelial and stromal cells were incubated with [35S]sulfate and [3H]glucosamine for 4 hr. The labeled glycosaminoglycans resulted from this incubation were isolated from the cell layer and the growth medium and further characterized. Both endothelial and stromal cell cultures synthesized a variety of sulfated glycosaminoglycans, with chondroitin 6-sulfate as the major product. Chondroitin 4-sulfate, dermatan sulfate, and heparan sulfate were present in smaller amounts. Keratan sulfate was produced only in minimal amounts. Both cell types also synthesized hyaluronic acid. The hyaluronic acid production in stromal cell strains derived from donors of different ages was similar. The demonstration that the endothelial cell strain derived from a 1-day-old baby contained more hyaluronic acid than cultures from older donors suggests a possible age-related phenomenon as seen in developing tissues.

Proteoglycan molecules in keratoconus corneas.

Proteoglycan molecules in keratoconus corneas were studied by immunohistochemical and electron microscopic histochemical methods. Compared with normal human control subjects, the staining intensity with monoclonal antibody 9-A-2 was enhanced in the stroma of scarred keratoconus corneas, whereas the intensity with antibody J-19 was reduced. The 9-A-2 experiment showed an increased immunoreactivity of dermatan sulfate proteoglycan epitopes, and the J-19 experiment indicated a decreased immunoreactivity of sulfated keratan sulfate epitopes. Uronic acid analyses were consistent with the 9-A-2 data. Electron microscopy performed after cuprolinic blue staining showed apparent accumulation of abnormally thick, chondroitinase ABC-sensitive, dermatan sulfate proteoglycan filaments in keratoconus corneas. Such filaments were especially prominent in scarred areas. In addition, Keratan sulfate proteoglycan filaments appeared to be less abundant than those found in normal control corneas. Similar alterations of both types of proteoglycan molecules were also seen and reported in scarred corneas. The similarity suggests that the proteoglycan abnormalities found in keratoconus corneas may be secondary, at least in part, to scarring.

Involvement of Sp1 elements in the promoter activity of genes affected in keratoconus.

PURPOSE: Keratoconus is a progressive disease that thins and scars the corneal stroma. In keratoconus corneas, levels of degradative enzymes, including lysosomal acid phosphatase (LAP) and cathepsin B, are elevated, and those of the inhibitors alpha1-proteinase inhibitor (alpha 1-PI) and alpha 2-macroglobulin (alpha 2-M) are reduced, especially in the epithelial layer. An increased expression of the transcription factor Sp1 was also demonstrated. The role of Sp1 in regulation of the genes affected in keratoconus was examined in this study. METHODS: DNA segments, containing 5'-flanking promoter sequences of the alpha 1-PI, LAP, cathepsin B, and alpha 2-M genes were ligated into the secreted alkaline phosphatase (SEAP) reporter gene vector. These constructs, along with the pSV beta-galactosidase control vector, were transfected into cultured human corneal epithelial and stromal cells and skin fibroblasts. Cotransfection with the Sp1 expression vector was performed in parallel. SEAP and beta-galactosidase enzyme activities were assayed. RESULTS: In corneal epithelial cells, as in stromal cells, alpha 1-PI promoter activity was suppressed by cotransfection of pPacSp1. The LAP, cathepsin B, and alpha 2-M promoters were functional in corneal cells, whereas activities of these promoters were much lower in skin fibroblasts. Cotransfection experiments indicated that the up- or downregulation of LAP, cathepsin B, and alpha 2-M observed in keratoconus-affected corneas was not mediated by Sp1. CONCLUSIONS: These results support the theory that the corneal epithelium, along with the stroma, is involved in keratoconus. An upstream role of Sp1 is indicated and the Sp1-mediated downregulation of the alpha 1-PI gene may be a key event in the disease development.

Expression of degradative enzymes and protease inhibitors in corneas with keratoconus.

PURPOSE: Keratoconus is characterized by thinning and scarring of the central region of the cornea. Previous research showed that, in corneas obtained from patients with keratoconus, lysosomal enzyme activities are elevated, whereas levels of protease inhibitors such as alpha1-proteinase inhibitor are reduced. This study was undertaken to examine further the expression of a spectrum of proteolytic enzymes and protease inhibitors. METHODS: Corneal buttons were collected from patients with keratoconus, healthy subjects, and patients with other corneal diseases. Immunohistochemical staining was performed on paraffin sections. Enzymatic assays and western blot analysis were carried out for cathepsins B and G. In addition, an in situ zymography procedure was used to examine the gelatin- and casein-digesting activities in corneas with keratoconus. RESULTS: An enhanced staining was found with antibodies to cathepsins B and G. Enzymatic assays and western blotting confirmed that the levels of these two enzymes were elevated in corneas with keratoconus. No alteration was noted with any of the matrix metalloproteinase (MMP) family members and other enzymes and inhibitors examined, although in situ zymography did indicate an increase in net gelatin- and casein-digesting activities in corneas with keratoconus. These activities were mostly abolished by inhibitors for serine and cysteine proteinases, but not by those for MMPs and aspartic proteinases. CONCLUSIONS: Levels of cathepsins B and G are increased in corneas with keratoconus. These enzymes may contribute to the heightened in situ gelatin- and casein-digesting activities, leading to abnormalities in keratoconus.

Lysosomal enzyme and inhibitor levels in the human trabecular meshwork.

PURPOSE: To examine in the human trabecular meshwork lysosomal enzymes and one inhibitor of serine proteases that actively participate in the degradation of macromolecules into low molecular weight constituents. METHODS: Using an avidin-biotin-peroxidase technique, lysosomal proteases and alpha 1-proteinase inhibitor were examined in the trabecular meshwork of 23 human eyes with donor ages ranging from 2 to 90 years. These eyes were categorized into three age groups (< or = 20, 21 to 49, and > or = 50 years). Histochemical staining for lysosomal hydrolases was also performed on frozen sections of 20 human eyes. The staining was analyzed by an image analyzer and the levels of lysosomal proteases were further measured by biochemical assays. RESULTS: The trabecular meshwork from all the eyes stained intensely against antibodies to cathepsins B and G and alpha 1-proteinase inhibitor. The staining for elastase was weaker but evident. Image analyses revealed that the staining intensity for each protease or inhibitor was similar in all age groups. The staining in the uveal meshwork appeared to be the strongest among all the trabecular meshwork regions. Biochemical assays of tissue extracts confirmed that the enzyme and inhibitor levels were comparable among the three donor age groups. Activities of two lysosomal hydrolases, acid phosphatase and acid esterase, were also found in trabecular meshwork cells of 20 eyes. No apparent difference in enzyme activities was found with increasing age, and variation related to region was not observed. CONCLUSIONS: This study demonstrated the age-independent distribution of a variety of lysosomal enzymes and a protease inhibitor in the human trabecular meshwork. The presence of these proteins suggests a possible role in the metabolic operation of the trabecular meshwork.

Corneal synthesis of alpha 1-proteinase inhibitor (alpha 1-antitrypsin).

PURPOSE: To determine if the cornea synthesizes alpha 1-proteinase inhibitor (alpha 1-antitrypsin). METHODS: Human corneas were placed in organ culture for 24 hours in the presence of 35S-methionine to radiolabel corneal proteins. Monoclonal antibodies were used to precipitate labeled alpha 1-proteinase inhibitor. The immunologically isolated inhibitor was electrophoresed on polyacrylamide gels and visualized by autoradiography or by staining for protein. Human corneas were also fixed with formalin and imbedded in paraffin. Sections were probed with 3H-labeled complementary DNA probes to the coding region of alpha 1-proteinase inhibitor. RESULTS: Metabolically labeled alpha 1-proteinase inhibitor was recovered from organ-cultured corneas and the cornea-conditioned medium. Specific messenger RNA was observed in the cornea by in situ hybridization most prominently in corneal epithelial cells. CONCLUSIONS: alpha 1-Proteinase inhibitor is synthesized and released by human corneal epithelial cells. These results indicate that the cornea has the ability to locally control degradation through synthesis of this inhibitor without total dependence on a supply of the inhibitor from the vascular system.

Alpha 2-macroglobulin is present in and synthesized by the cornea.

PURPOSE: The purposes of this study were to determine whether the proteinase inhibitor alpha 2-macroglobulin is present in the cornea, and, if so, where it is located, and whether it is synthesized by the cornea, and, if so, where it is being synthesized. METHODS: alpha 2-Macroglobulin was immunolocalized using a double antibody technique and quantified by immunodot blot assays, and its identity was confirmed by Western blot analysis. Corneal synthesis of this inhibitor was determined by immunoprecipitation of extracts from corneas incubated in organ culture with 35S-methionine. mRNA was localized by in situ hybridization of 3H-labeled cDNA to the inhibitor. RESULTS: alpha 2-Macroglobulin was localized in the epithelial, endothelial, and stromal cells. It was also found in the stromal extracellular matrix. When extracts of the epithelium, stroma, and Descemet's membrane-endothelium were analyzed by Western blot, an immunoreactive band for this inhibitor was detected in all extracts. This band comigrated with the alpha 2-macroglobulin form isolated from plasma. Metabolically labeled inhibitor was immunoprecipitated from the stromal layer but not from the epithelial or endothelial layer. However, when examined by in situ hybridization, mRNA was localized to epithelial and endothelial cells in addition to stromal keratocytes. CONCLUSIONS: Because alpha 2-macroglobulin has the ability to inhibit a wide range of proteinases, it is probable that this inhibitor plays an important role in protecting the cornea from damage caused by proteinases. This includes proteinases synthesized by the cornea and those released from inflammatory cells and invading organisms.

Loss of cell-matrix cohesiveness after phagocytosis by trabecular meshwork cells.

PURPOSE: To investigate the response of trabecular meshwork cells to phagocytic events. METHODS: Cultured bovine trabecular meshwork cells were established and exposed to latex microspheres for 40 to 44 hours. After phagocytosis, the cohesiveness of cells to their underlying matrix was measured by the susceptibility to trypsin, as indicated by the time needed to be liberated from culture plates. The amounts of two cell attachment proteins, fibronectin and laminin, in both the phagocytically challenged and the control cultures were measured at various postphagocytosis time points with an enzyme-linked immunosorbent assay. The fibronectin and laminin network was visualized with immunostaining. The mRNA levels were analyzed by Northern blot. Zymography using gelatin-containing gels was also performed to examine the gelatinase activities. RESULTS: Compared with controls, cells in phagocytically challenged cultures were more sensitive to trypsin. At the 4- and 8-hour postphagocytosis time points, the trypsinization time needed to suspend cells from tissue culture plates was significantly shorter for phagocytically challenged cells. Also, at these two time points, reduced amounts of fibronectin and laminin, as well as disruption of the fibronectin-laminin network, were observed in the phagocytically challenged trabecular meshwork cultures. The mRNA level for fibronectin was reduced, and a slightly increased gelatinase activity was noted. The fibronectin and laminin levels returned to normal by 24 hours. CONCLUSIONS: Results suggest that after phagocytosis, trabecular meshwork cells exhibit a short-term loss in cell-matrix cohesiveness. Such a loss may be related to diminished levels of cell attachment proteins.

Effects of rho-associated protein kinase inhibitor Y-27632 on intraocular pressure and outflow facility.

PURPOSE: To elucidate the roles of Rho-associated protein kinase (ROCK) in regulating intraocular pressure (IOP) and outflow facility in the rabbit eye. METHODS: A specific ROCK inhibitor Y-27632 was used. The IOP, the outflow facility, and the pupil diameter were determined before and after the topical, intracameral, or intravitreal administration of Y-27632 in rabbits. Western blot analysis was used to identify specific ROCK isoform in human trabecular meshwork (TM) cells and bovine ciliary muscle (CM) tissues. The cell morphology and distribution of actin filaments and vinculin in TM cells were studied by cell biology techniques. Carbachol (Cch)-induced contraction of isolated bovine CM strips after administration of Y-27632 was measured in a perfusion chamber. RESULTS: In rabbit eyes, administration of Y-27632 resulted in a significant decrease in IOP in a dose-dependent manner. An increase of the outflow facility and pupil size dilation was also observed in Y-27632-treated eyes. Western blot analysis revealed the presence of p160ROCK in human TM cells and bovine CM tissues. In cultured human TM cells, exposure to Y-27632 caused retraction and rounding of cell bodies as well as disruption of actin bundles and impairment of focal adhesion formation. Y-27632 in addition inhibited Cch-induced contraction of isolated bovine CM strips. CONCLUSIONS: Administration of Y-27632 caused a reduction in IOP and an increase in the outflow facility. The in vitro experiments suggest that the IOP-lowering effects of Y-27632 may be related to the altered cellular behavior of TM cells and relaxation of CM contraction. These studies suggest that ROCK inhibitors may have great potential to be developed for treatment of glaucoma and other ocular diseases.