Effects of Y27632 on keratinocyte procurement and wound healing.

A number of Rho-kinase inhibitors have been developed for various clinical applications. We examined the effects of the Rho-kinase inhibitor Y27632 on keratinocyte proliferation and migration, and found that it promoted primary human keratinocyte proliferation and migration in both monolayer and skin explant cultures. In addition, topical application of Y27632 enhanced cutaneous wound closure in the majority of wounds in mice. The growth and migration effects of Y27632 appeared to be specific to keratinocytes compared with dermal fibroblasts, and required intact Jun kinase function. Y27632 seems to be a promising agent for keratinocyte procurement and wounding healing.

Higher densities of distributed pheromone sources provide disruption of codling moth (Lepidoptera: Tortricidae) superior to that of lower densities of clumped sources.

Field experiments quantified the effect of synthetic pheromone release-site density and distribution on 1) orientational disruption of male codling moths, Cydia pomonella (L.) (Lepidoptera: Tortricidae), to pheromone-baited traps; and 2) fruit injury. A clustering test varied pheromone release-site density from 0 to 1,000 Isomate-C Plus dispensers per ha while maintaining the total number of dispensers at 1,000. Percentage of orientational disruption of pheromone-baited traps increased significantly as a function of increasing density of release sites. Fruit injury decreased as the density of release sites increased and was lowest in plots treated with Isomate-C Plus dispensers distributed as 1,000 point sources per ha. We also manipulated point source density of 0.1-ml paraffin-wax drops containing 5% codlemone [(E,E)-8,10-dodecadien-1-ol], and thus the total amount of pheromone deployed per hectare. The percentage of disruption of traps baited with either 1.0- or 0.1-mg codlemone lures increased with increasing density of wax drops deployed. Both trapping and field observations confirmed that wax drops were attractive to male codling moths, suggesting that disruption was mediated by competitive attraction. Development of dispensers that can be mechanically applied at high densities has potential to improve the efficacy and economics of codling moth disruption at high population densities.

Identification and isolation of differentially expressed genes from very small tissue samples.

Identification of differentially expressed genes from tissue samples weighing only a few milligrams has remained a major challenge. Here, we describe a novel and simple strategy that uses standard molecular biology equipment and commercially available kits. The approach combines isolation of total RNA by silica-gel binding, reverse transcription using anchored modified, 5' end enhancers oligonucleotides, exponential amplification of the single-stranded cDNA and hybridization to high-density cDNA filter arrays. The method was tested by comparing genes expressed on freshly isolated human trabecular meshwork tissue with those expressed in corresponding primary cells at third passage. Validation was achieved by using two biological properties: (i) hybridization, to identify the differentially expressed genes, and (ii) PCR amplification, to confirm their distinct expression. The strategy presented allows the identification of differentially expressed genes and/or uncharacterized expressed sequence tags (ESTs) in very small tissue samples, including those from clinical specimens.

Morphology of the trabecular meshwork and inner-wall endothelium after cationized ferritin perfusion in the monkey eye.

Nine eyes of five cynomolgus monkeys were perfused through the anterior chamber with cationized ferritin (CF) at normal and increased intraocular pressure. After perfusion with glutaraldehyde, the morphologic appearance of the endothelial lining of Schlemm's canal (SC) and of the adjacent regions was analyzed using tangential and sagittal serial sections. The CF, which binds to negatively charged surfaces, was observed to be adherent to the free surfaces of trabecular cell membranes and to accumulate in the cribriform layer underlining the endothelial lining of SC. Tangential sections of the inner-wall endothelium demonstrated that separations of the adjacent cell membranes occur between the tight junctions forming openings or lacunae and bent, tunnel-like channels that represent continuous paracellular pathways. Complete staining of these inner-wall paracellular pathways with CF were observed indicating that the adjoining membranes are negatively charged and that the perfused fluid had passed through these intercellular channels. These paracellular pathways appeared enlarged and were more easily identified at elevated perfusion pressure. In general, intracytoplasmic vacuoles demonstrated heavy staining with CF on their luminal surface but only faint staining on the adluminal (cribriform-facing) surface. Apparent giant vacuoles were observed to be often not real intracellular vacuoles but rather dilatations of the paracellular spaces. This study demonstrates that there are paracellular routes through the inner-wall endothelium by which high molecular-weight substances such as ferritin and macrophages can leave the anterior chamber. Probably there are both transcytoplasmic and paracellular mechanisms of aqueous outflow that may vary under different conditions of pressure or flow.

Influence of intraocular pressure on aqueous outflow facility in enucleated eyes of different mammals.

Freshly enucleated monkey, calf, and human eyes were quantitatively perfused with mock aqueous humor through the anterior chamber by a constant pressure technique. After baseline perfusion at 15 mm Hg, intraocular pressure was raised to 45 mm Hg and later reduced back to 15 mm Hg. Calf and human (both adult and infant) eyes had lower outflow facilities at 45 than at 15 mm Hg. However, four types of monkey eyes did not show decreased facility of outflow at elevated perfusion pressure, and after return of pressure to 15 mm Hg, facility of outflow actually increased compared to baseline, unlike both calf and human eyes. The results indicate that there are significant differences in the response of enucleated mammalian eyes to an elevation in perfusion pressure. Factors other than, or in addition to, collapse of Schlemm's canal may be important in the pathogenesis of the pressure-induced decrease in outflow facility found in human eyes.

Characterization of gene expression in human trabecular meshwork using single-pass sequencing of 1060 clones.

PURPOSE: To study the gene expression profile of the human trabecular meshwork (HTM). METHODS: A polymerase chain reaction (PCR)-amplified cDNA library was constructed using RNA from the TM of a 67-year-old normal, perfused human eye. A total of 1060 clones were randomly selected for sequencing of one end. These sequences were searched against nonredundant GenBank and dbEST databases for similarity comparison by using a FASTA file and the BLASTcl3 program. Relative expression patterns of those clones that matched other expressed sequence tags (ESTs) were determined using the National Center for Biotechnology Information (NCBI) Unique Human Gene Sequence Collection (UniGene) database. RESULTS: Of the 1060 clones analyzed, 519 (48.9%) had sequences identical with known genes, 125 (11.8%) matched ESTs, and 189 (17.8%) did not match any database sequences. Of the remaining clones, 31 (3%) corresponded to mitochondrial transcripts and 196 (18.5%) to repetitive and noninformative sequences. It is notable that some of the genes highly represented in this library are not ubiquitously expressed in other tissues, which suggests a potentially important role in the HTM. As evidence for the presence of true novel genes in the library, one of the clones was fully sequenced. This clone comprised a complete open reading frame of 966 nucleotides, and its deduced amino acid sequence corresponded to a protein 33% similar to the MAS-related G-protein-coupled receptor. CONCLUSIONS: The identification of the more highly expressed genes in HTM and the discovery of novel genes expressed in this tissue provides basic information for further research on the physiology of the TM and for the identification of glaucoma candidate genes.

Anterior and posterior axial lens displacement and human aqueous outflow facility.

We studied the effect of anterior and posterior axial crystalline lens displacement (and thereby ciliary tone) on the aqueous humor outflow facility in enucleated human eyes. After attaching a reversible footplate plunger to the anterior lens capsule with cyanoacrylate, the lens was placed in one of three positions: "neutral baseline," posterior displacement (2.5 mm), or anterior displacement (2.0 mm). In seven pairs of eyes, the mean "neutral baseline" was not statistically different from the control eye, but anterior lens displacement decreased outflow facility "C" by 0.14 (36%) (P less than 0.0001), and posterior displacement increased "C" by 0.18 (50%) (P less than 0.01). Anterior or posterior lens displacement after complete ciliary body detachment produced no effect on outflow facility in two pairs. Histologic correlation studies demonstrated narrowing of the intertrabecular spaces and Schlemm's canal in the eyes fixed in anterior lens displacement, and widening of the same structures in the eyes fixed in posterior lens displacement. The lens-zonular-ciliotrabecular force vectors are responsible for the compression or decompression of the meshwork and Schlemm's canal in this model, with compression decreasing, and decompression increasing aqueous humor outflow facility.

Ethacrynic acid induces reversible shape and cytoskeletal changes in cultured cells.

Cell cultures derived from trabecular meshworks of human and bovine eyes and from bovine vascular endothelia were incubated at 37 degrees C for 1 hr with ethacrynic acid (ECA, 0.1-0.5 mmol/l) dissolved in culture medium. At 2 hr after the initial exposure, ECA at concentrations up to 0.4 mmol/l induced a reversible alteration in cell shape in all three cell types that was coincident with a change in the staining pattern of major cytoskeletal components including actin, alpha-actinin, vinculin, and vimentin. Distinct progressive alterations in beta-tubulin also occurred, with initial changes observed 10 min after ECA exposure. The ECA-induced changes in tubulin were blocked in part by preincubation with taxol (which stabilizes the microtubule structure), but they appeared to differ from those occurring with nocodazole (which interferes with tubulin assembly). These results suggest the possibility that ECA-induced increases in outflow facility may be mediated by alterations in the cytoskeletons of outflow pathway cells.

Effect of age on superoxide dismutase activity of human trabecular meshwork.

PURPOSE: It has been hypothesized that accelerated aging of the trabecular meshwork, perhaps because of oxidative damage, is involved in the pathogenesis of primary open angle glaucoma. The authors sought to evaluate the effect of donor age on the specific activity of superoxide dismutase and catalase in normal fresh human cadaver trabecular meshwork. METHODS: Total superoxide dismutase and catalase were assayed in tissue extracts generated from fresh human trabecular meshwork. Cadaver tissue was obtained from 19 donors (18 paired) of a wide age range (30 to 91 years). The assays were performed within 6 hours of enucleation and within 36 hours of donor death. Enzyme-specific activities were calculated using protein concentration of the extract as the denominator. RESULTS: Multiple regression analysis modeled with age and time from death until the beginning of the experiment was performed. The specific activity of superoxide dismutase declined with age (P = 0.00022; r2 = 0.67). There was no effect of age on catalase specific activity (P = 0.24; r2 = 0.16). The time from donor death until the beginning of the experiment was not a significant factor (P > 0.28). CONCLUSIONS: The specific activity of superoxide dismutase, but not catalase, demonstrates an age-dependent decline in normal cadaver human trabecular meshwork. The potential role of superoxide dismutase in primary open angle glaucoma, a disorder of the aging trabecular meshwork, warrants further investigation.

Genes upregulated in the human trabecular meshwork in response to elevated intraocular pressure.

PURPOSE: To identify genes upregulated in perfused, intact human trabecular meshwork (TM) in response to elevated intraocular pressure (IOP). METHOD: Two pairs of anterior segments of normal human eyes from postmortem donors were placed in culture and perfused 24 hours at constant flow (3 microl/min). After reaching baseline, the flow of one eye from each pair was raised to obtain an incremental pressure (deltaP) of 50 mm Hg for 6 hours. The anterior segments were then quickly frozen in liquid nitrogen, and their TMs were dissected for RNA extraction. SMART cDNA libraries were generated from control and high-pressure human TM RNAs and hybridized to sets of identical high-density cDNA gene arrays. These arrays contained 18,376 human expressed sequence tags (ESTs), corresponding to both characterized and unknown genes. Differentially expressed genes were identified by different-intensity hybridization signals and confirmed by semi-quantitative polymerase chain reaction. RESULTS: Eleven genes were found to be consistently upregulated in the human TM by elevated IOP: interleukin-6, preprotachykinin-1, secretogranin-II, cathepsin-L, stromelysin-1, thymosin-beta4, alpha-tubulin, alphaB-crystallin, glyceraldehyde-3-phosphate dehydrogenase, metallothionein and Cu/Zn superoxide dismutase. The products of these genes are involved in vascular permeability, secretion, extracellular matrix remodeling, cytoskeleton reorganization, and reactive oxygen species scavenging. CONCLUSIONS: Elevated IOP induced specific upregulation of 11 physiologically relevant genes. On the basis of their known activities, the products of each of these genes might predict homeostatic mechanisms similar to those involved in the regulation of blood vessel permeability. We hypothesize that similar mechanisms might be involved in regulating flow through Schlemm's Canal endothelium.

Metabolism of calf trabecular (reticular) meshwork.

It has been found that calf eyes are an excellent source of trabecular meshwork tissue for biochemical studies. Homogenates of pooled meshwork were centrifuged at 27K X g and 1.5K X g. The high-speed supernatants produced lactate at 0.35 mumole/min/gm tissue in the presence of hexokinase-saturating concentrations of glucose (10 mM) at pH 7. The optimum pH was 7.6. In the absence of ammonia, lactate could be produced from fructose 1,6-diphosphate but not from glucose or glucose 6-phosphate. The optimum ammonia concentration was 1 to 2 mM. Lactate was produced at an even greater rate from fructose, but only poorly from sorbitol or galactose (all at 10 mM). The activity of hexokinase, glucose 6-phosphate dehydrogenase and UDPG dehydrogenase was measured. Fructokinase could not be detected. The low-speed supernatant readily oxidized succinate, malate, and glutamate at about 0.012 muAtO/min/gm tissue. The oxidative rate in vivo is estimated to be about one third of this. These results demonstrate that knowledge of the normal metabolism of calf trabecular meshwork may be obtained with relative ease, with possible important implications for understanding the disease of glaucoma.

Glucose 6-phosphate dehydrogenase of calf trabecular meshwork.

Activities of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of calf trabecular meshwork were measured and found to be 0.23 and 0.47 mumole/min/g tissue, respectively. Glucose 6-phosphate dehydrogenase was purified 450-fold with a yield of 91% by anion exchange chromatography and 2',5'-ADP agarose affinity chromatography. It was activated by Ca2+, Mg2+, and Mn2+. It was deactivated by p-chloromecuribenzoate, p-chloromercuribenzene sulfonate, and iodoacetamide, but this deactivation could be prevented by pretreatment with cysteine or glutathione. Its rate was regulated by the NADPH/NADP+ ratio, being maximal at a ratio of 0, and negligible at a ratio of 10. At the physiological ratio of 5, its rate was approximately half maximal. On disc gel electrophoresis of both the crude and purified enzyme, seven bands of glucose 6-phosphate dehydrogenase activity could be seen. The isozyme pattern was similar to that of calf retina, but different from that of calf liver. These data suggest that trabecular meshwork is well supplied with the capacity to generate NADPH. Typical demands for NADPH may be to detoxify H2O2 and/or organic peroxides through the glutathione peroxidase/glutathione reductase system, and both generating and removing products of the "killing reaction" during phagocytosis.

Localization of smooth muscle and nonmuscle actin isoforms in the human aqueous outflow pathway.

alpha-Smooth muscle actin is the isoform of actin restricted to vascular smooth muscle, pericytes, myofibroblasts and, certain other cells that are of myoid origin. We investigated the distribution of alpha-smooth muscle actin and nonmuscle specific filamentous actin in the human aqueous outflow system by immunohistochemical methods. Filamentous actin was observed in all cellular constituents of the outflow pathway, while distribution of alpha-smooth muscle actin was restricted to the ciliary muscle, to specific cells throughout the trabecular meshwork, and to cells adjacent to the outer wall and the collector channels. The ciliary muscle extended deep into the corneoscleral meshwork, far anterior to the scleral spur. These findings agree with our previous study localizing the distribution of smooth muscle myosin in the human aqueous outflow pathway. Although functionality of the immunoreactive cells needs to be demonstrated, our data show that a potentially contractile apparatus exists in a subpopulation of trabecular meshwork cells and in certain cells of the more distal components of the outflow system.

Nonsulfhydryl-reactive phenoxyacetic acids increase aqueous humor outflow facility.

PURPOSE: The phenoxyacetic acid, ethacrynic acid (ECA), has potential use in glaucoma therapy because it acts to increase aqueous outflow in vivo and in vitro. In human trabecular meshwork (HTM) cell culture, ECA acts to change cell shape and attachment, effects that have been correlated with microtubule (MT) alterations and chemical sulfhydryl (SH) reactivity. To further explore these actions, we evaluated two non-SH reactive phenoxyacetic acids, inadcrinone and ticrynafen, and the MT-disrupting drug vinblastine. METHODS: Excised bovine and porcine eyes were perfused and outflow facility measured. Calf pulmonary artery endothelial and HTM cells were grown in culture and cytoskeletal effects evaluated after drug treatment. RESULTS: Indacrinone, ticrynafen, and vinblastine all caused an increase in outflow facility. In contrast with ECA, the outflow effects of indacrinone and ticrynafen were not blocked by excess cysteine. Although indacrinone and ticrynafen produced changes in cell shape in vitro, the beta-tubulin staining pattern of treated cells was not altered. Vinblastine caused cell shape change and the expected MT disruption. CONCLUSIONS: Phenoxyacetic acids can increase aqueous outflow facility and alter HTM cell shape and attachment in vitro by a non-SH, non-MT mechanism (which is probably shared also by ECA). These findings suggest the possibility of a broader class of glaucoma drugs that may be directed at the HTM. An understanding of the cellular target for these drugs has implications both for potential glaucoma therapy and for the cytoskeletal mechanisms involved in normal outflow function.

Glutathione in calf trabecular meshwork and its relation to aqueous humor outflow facility.

Previous studies have shown that sulfhydryl reagents can alter the facility of aqueous humor outflow but little is known about the sulfhydryl constituents of the aqueous outflow system or the effect of oxidants upon outflow facility. In the present study the concentration of glutathione (GSH) was measured in excised calf trabecular meshwork (TM) and found to be 0.40 mu mol/g wet wt (0.027 mu mol/mg protein). Oxidized glutathione was not detectable in the tissue. TM was found to have significant hexose monophosphate shunt activity as determined by measurement of the oxidation of 14C-1 and 14C-6-labeled glucose in tissue homogenates. The concentration of GSH in TM of enucleated calf eyes could be totally depleted by infusion of medium containing both diamide, which is an oxidant of GSH, and 1,3bis(2-chlorethyl)-1-nitrosourea (BCNU), which is an inhibitor of the enzyme glutathione reductase. The depletion of GSH was found to have no effect on the facility of aqueous outflow. Experiments were also done in which normal and TM GSH-depleted eyes were perfused with medium containing H202. Exposure to H202 produced no effect on outflow facility in the normal eyes but caused a 33% decrease in facility in eyes with the GSH-depleted TM. The results indicate that GSH may not participate directly in regulating aqueous humor outflow but is able to protect TM against H202-induced oxidative damage that would otherwise lead to a decrease in outflow facility.

Morphology and function of the aqueous outflow system in monkey eyes perfused with sulfhydryl reagents.

The aqueous outflow system from anterior chamber to Schlemm's canal was examined by electron microscopy in pairs of enucleated macaque and baboon eyes, perfused via the anterior chamber with mock aqueous humor in one eye and the same fluid with added iodoacetamide (IA) or N-ethyl maleimide (NEM) in the other eye. Many details of the electron micrographs were analyzed in a masked manner using a digitizing bit pad and computer, and also using visual evaluation. Both IA and NEM increased aqueous humor outflow facility, but the morphologic changes induced by IA were quantitatively different from those induced by NEM. Intercellular junctions were not affected by IA, but were disrupted by NEM (P less than 0.01). Vacuoles in the endothelial lining of Schlemm's canal tended to increase in area, but not in number, under the influence of IA, whereas they were not so affected by NEM. No loss of extracellular material was observed in either IA- or NEM-treated eyes. The results indicate that the chemical status of cellular-SH groups may influence aqueous humor outflow facility at multiple sites in the outflow pathway.

Identification of isoenzyme C as the principal carbonic anhydrase in human ciliary processes.

Carbonic anhydrase was extracted from the excised processes of the ciliary bodies of 10 pairs of enucleated human eyes, and the isoenzyme composition was assayed by measuring the degree of inhibition produced by acetazolamide at two standard concentrations. Also, the effect of incubating with iodoacetate was determined in two pairs of these eyes. Only isoenzyme C was detected. Accordingly, it seems that differences in reduction of intraocular pressure that are common among patients treated for glaucoma with systemic carbonic anhydrase inhibitors, despite uniform serum concentrations, are not attributable to interindividual variation of carbonic anhydrase isoenzymes in the ciliary processes.

Microtubule disruption leads to cellular contraction in human trabecular meshwork cells.

PURPOSE: To determine whether microtubule- and actin-altering drugs, which have been shown to increase aqueous humor outflow, cause cellular contraction in human trabecular meshwork (HTM) cells. METHODS: HTM cells were plated in culture dishes containing a polymerized deformable silicone substrate. After 48 hours, the dishes were placed on an inverted microscope and treated with ethacrynic acid, colchicine, vinblastine, cytochalasin B, or 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and then recorded on videotape for 15 minutes. An increase in silicone substrate wrinkle size and/or number indicated a contraction. Sham controls were used. RESULTS: Cellular contraction was observed with ethacrynic acid, colchicine, and vinblastine in the 10(-5) to 10(-4) M dosage range. Pretreatment with H-7 blocked these effects. Cytochalasin B did not produce cellular contraction. CONCLUSIONS: Microtubule disruption causes cellular contraction in HTM cells, and this effect depends on an intact actin cytoskeleton network. Contraction of trabecular meshwork cells in response to various stimuli is an attractive hypothesis for possible homeostatic mechanisms in the outflow pathway, and this may serve as a focus for novel glaucoma drug development.

Hydraulic pressure stimulates adenosine 3',5'-cyclic monophosphate accumulation in endothelial cells from Schlemm's canal.

PURPOSE: Fluid flow across various endothelia results in a variety of intracellular and extracellular adaptations. In the living eye, aqueous humor flows across the surface of endothelial cells on trabecular meshwork (TM) beams and in the juxtacanalicular tissue and through or between a continuous monolayer of endothelial cells that line Schlemm's canal (SC). This study was undertaken to test the hypothesis that fluid flow induces biochemical changes in the endothelial cells of the outflow pathway that may modify outflow resistance. METHODS: Trabecular meshwork and SC cells isolated from the outflow pathway of human cadaveric eyes were seeded onto porous filters, placed in Ussing-type chambers, and subjected to fluid flow driven by a pressure head of 15 mm Hg on their apical surface. Cell lysates were prepared and analyzed for adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. Barrier function of cell monolayers was examined using transendothelial electrical resistance measurements. RESULTS: Three different SC cell strains in 14 independent experiments responded with at least a threefold increase in cAMP that was both time and pressure dependent. Conversely, flow-treated TM cells failed to respond in six independent experiments in which five different TM cell strains were used. Electrical resistance across cell monolayers positively correlated with cAMP accumulation and was calcium sensitive. CONCLUSIONS: cAMP signaling is affected by pressure differentials across SC cell monolayers and provides evidence for the participation of SC cells in the regulation of aqueous outflow.

Phosphofructokinase of calf trabecular meshwork.

The activity of phosphofructokinase (PFK), a key regulatory enzyme of glycolysis, has been measured in the 27,000 X g supernatant of homogenates prepared from excised calf trabecular meshwork. The enzyme required NH4+, both at pH 8.5 and pH 7.2. This requirement was not relieved by K+ or AMP. At pH 7.2 and ATP levels of 0.1 to 2.5 mM, PFK was completely inactive in the absence of added AMP or NH4+. PFK was only weakly activated by 0.5 mM AMP or by 5 mM NH4+, but in the presence of both AMP and NH4+, PFK was highly active up to 1 mM ATP. At pH 8.5 and ATP levels of 0.1-12.5 mM, PFK was weakly active in the absence of added NH4+, with or without AMP. With the addition of 5 mM NH4+, PFK was highly active up to 2.5 mM ATP, while AMP was largely without effect. Concentrations of NH4+ as low as 0.03 mM stimulated PFK activity to 20% of maximal, yet the maximum was not reached until NH4+ levels were 10-30 mM. The activation of PFK by AMP and its inhibition by ATP is profoundly modified by pH. In contrast, the requirement for NH4+ is unaffected. This requirement suggests a regulatory role for ammonium ion in controlling the rate of glycolysis in trabecular meshwork. The concentration of ammonium in calf aqueous humor was found to be 0.18 mM, which is in the right range to have an effect.