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PubMed2006

Caldesmon transgene expression disrupts focal adhesions in HTM cells and increases outflow facility in organ-cultured human and monkey anterior segments.

Cytoskeleton modulating compounds have been shown to lower intraocular pressure (IOP) and increase outflow facility. Caldesmon is one protein that is involved in the regulation of actin stress fiber formation. The effects of rat non-muscle caldesmon (Cald) gene over-expression on focal adhesions in human trabecular meshwork (HTM) cells and on outflow facility in organ-cultured human and monkey anterior segments were determined. Treatment of HTM cells with adenovirus-delivered caldesmon (AdCaldGFP) resulted in characteristic changes in the actin cytoskeleton and matrix adhesions within 24-48 hr post-transduction. Stress fibers gradually disappeared and novel actin structures were formed (see manuscript by Grosheva et al., this issue). In cells with disrupted stress fibers, vinculin-containing focal adhesions were also disrupted. In organ-cultured anterior segments, baseline outflow facility (microl min-1 mmHg-1) for all anterior segments averaged (mean+/-sem): human, 0.19+/-0.03 (n=12); monkey, 0.36+/-0.02 (n=19). In human anterior segments, transduction with 10(7) plaque forming units of AdGFPCald increased outflow facility by 43+/-21% (p<or=0.11, n=6) at 66 hr compared to baseline and corrected for the changes in outflow facility of the contralateral vehicle treated segment. Using the same time point, i.e. 2-3 days after injection, outflow facility in monkey anterior segments, transduced with 1.5x10(7) plaque forming units of AdGFPCald was increased by 35+/-18%, p<0.2, n=10 compared to baseline and corrected for the change in outflow facility in the contralateral AdGFP treated segment. Combining human (66 hr) and monkey (2-3 days) data, outflow facility was increased by 38+/-13%, p<0.02, n=16. Additional analysis of maximum responses in monkey anterior segments from 1 to 6 days after transduction showed outflow facility was increased by 66+/-18%, p<0.01, n=10. Caldesmon over-expression, which relaxes cultured HTM cells and disrupts their actin cytoskeleton and cell-matrix adhesions, also appears to increase outflow facility in organ-cultured human and monkey anterior segments. This suggests that over-expression of the caldesmon gene in the TM may be an effective approach for the gene therapy of glaucoma.

Actins↗
PubMed2002

Effects of elevated intraocular pressure on outflow facility and TIGR/MYOC expression in perfused human anterior segments.

PURPOSE: To investigate the effects of high intraocular pressure (h-IOP) on TIGR/MYOC expression, extracellular matrix (ECM) deposition, and outflow facility (C) in perfused human anterior segment cultures. METHODS: Anterior segments of 31 pairs of normal human eyes from postmortem donors were perfused at constant flow (3 microl/min). After reaching stable baseline, the flow of one eye from each of 31 pairs was raised to obtain a continuous pressure of 60 to 70 mm Hg for a period of 1 hour (3 pairs), 6 hours (10 pairs), 24 hours (2 pairs), 48 hours (3 pairs), and 7 days (13 pairs). Sixteen of these pairs were used to study trabecular meshwork expression of TIGR/MYOC and stromelysin by Northern blot analysis hybridization. Nine pairs (1 pair each at h-IOP for 1, 6, and 48 hours and 6 pairs at 7 days) were fixed at pressure for analysis by electron microscopy. Eyes selected for C measurements fulfilled the inclusion criteria of C0 values between 0.06 and 0.4, intact RNA recovery and normal light microscopy morphology. Percent change of facility from the baseline (C/C0) was computed at 6 and 24 hours and 2, 4, and 7 days from the long-term perfusion experiments (n = 9 h-IOP, n = 8 controls). RESULTS: No induction of TIGR/MYOC expression was observed after h-IOP for 1 and 6 h. A slight induction was seen after 24 and 48 hours. At 7 days, the treated eye from 4 of 5 pairs showed a clear induction, which was very pronounced in one of the pairs. In contrast, stromelysin expression was induced at 6 hours and not at 7 days. Morphometric electron microscopy after 7 days showed no significant difference in the amounts of fine fibrillar material or plaque material in the juxtacanalicular (JCT) region. The percent increase of C of the treated eye at 6 hours was 11.0% +/- 4.6% compared with 3.7% +/- 3.8% in the control eyes (P = 0.26). However, after longer time periods, the facility of the h-IOP eyes increased, whereas that of the contralateral eyes remained unchanged. This difference reached peak, significant values at 4 days (32.9% +/- 8.4% versus 7.4% +/- 7.6%, respectively; P = 0.04) and decreased to 8.9% +/- 7.9% versus 1.1% +/- 12.7% (P = 0.6) at 7 days. CONCLUSIONS: Elevated IOP appears to cause a decrease in outflow pathway resistance at 1 to 4 days, and this effect seems to disappear with further time. In contrast, induction of TIGR/MYOC appears to be strongest at 7 days. We speculate that this induction pattern might indicate a stress-related, rather than a possible homeostatic, role for the TIGR/MYOC protein.

Adult↗
PubMed2005

Blebbistatin, a novel inhibitor of myosin II ATPase activity, increases aqueous humor outflow facility in perfused enucleated porcine eyes.

PURPOSE: To investigate the specific role of myosin II, a critical biochemical determinant of cellular contraction, in modulation of aqueous humor outflow facility through the trabecular meshwork (TM) pathway. METHODS: Expression of the nonmuscle myosin II heavy chains (IIA, IIB, and IIC) in human TM and ciliary body (CB) cells was determined by RT-PCR analyses. The effects of inhibition of myosin II on cell morphology, actomyosin organization, and cell adhesions were evaluated in porcine TM and CB cells treated with blebbistatin, a cell-permeable, specific inhibitor of myosin II adenosine triphosphatase (ATPase) activity. Changes in aqueous humor outflow facility were determined in enucleated porcine eyes by using a constant-pressure Grant perfusion model system. Ultrastructural integrity of the outflow pathway in drug-perfused eyes was analyzed by transmission electron microscopy. RESULTS: Expression of nonmuscle myosin IIA and IIB was confirmed in both human TM and CB cells. Confluent cultures of primary porcine TM and CB cells treated with blebbistatin in the presence of serum revealed dose (10-200 microM)-dependent changes in cell morphology, decreases in actin stress fiber content and in focal adhesions and adherens junctions. These changes were found to be reversible within 24 hours of drug withdrawal from the cell culture media. Blebbistatin did not affect the status of myosin light chain phosphorylation in TM cells. Perfusion of enucleated porcine eyes for 5 hours with 100 and 200 microM blebbistatin produced a significant increase (P < 0.01, n = 7) in aqueous outflow facility (53% and 64%, respectively) from the baseline facility, compared with a 21% facility increase in sham control specimens. The integrity of the inner wall of aqueous plexi in drug-perfused porcine eyes was found to be intact, and TM cell morphology appeared to be similar to that noted in sham-treated eyes. CONCLUSIONS: These data demonstrate that selective inhibition of myosin II in the aqueous humor outflow pathway leads to increased aqueous outflow facility, suggesting a critical role for myosin II in the regulation of aqueous humor outflow facility. This study also suggests myosin II as a potential therapeutic target for lowering intraocular pressure in patients with glaucoma.

Adenosine Triphosphatases↗
PubMed2002

Preservation of aqueous outflow facility after second-generation FIV vector-mediated expression of marker genes in anterior segments of human eyes.

PURPOSE: Feline immunodeficiency virus (FIV)-based lentiviral vectors produce effective genetic modification of the trabecular meshwork (TM) of human eyes in organ-perfusion culture, resulting in high-level expression of a beta-galactosidase marker gene (lacZ) without loss of TM cellularity or architecture. However, effects on aqueous outflow physiology have not been determined, and the ability to monitor FIV vector transgene expression in living TM in situ has not been established. In the current study, transgene expression and outflow facility were evaluated in perfused human anterior segments after FIV vector transduction of lacZ or of a marker gene that can be monitored noninvasively, enhanced green fluorescent protein (eGFP). METHODS: Second-generation FIV vectors were made with a protocol for scaled-up production that requires 10 times less input DNA and allows simplified concentration. One vector encodes beta-galactosidase (vector CT26), and the other (bicistronic) encodes eGFP and neomycin phosphotransferase (vector GiNWF). Three pairs of eyes were injected with 1 x 10(8) transducing units (TU) of CT26 in the right eye and with a control (mock lacZ) vector in the left eye. Three others were injected with 1 x 10(8) TU GiNWF in the right eye only, with the left eye serving as an uninjected control. Intraocular pressure was recorded and transduction efficiency was determined. RESULTS: The modified protocol produced high-titer FIV vectors, and coordinate expression of marker genes was observed with the bicistronic vector. In human eyes, the eGFP and lacZ vectors transduced 79% +/- 15% and 82% +/- 4% of TM cells, respectively, without cell loss compared with control eyes. Transduction and marker gene expression caused a transient decrease of outflow facility (30% +/- 22%, P = 0.02), which resolved after 48 to 72 hours. CONCLUSIONS: FIV vectors produce high-level expression of eGFP in the TM of the cultured human eye, with transduction efficiency similar to that obtained with beta-galactosidase vectors. Transduction and expression of these marker genes results in small and transient changes in outflow facility, suggesting suitability of this class of vectors for glaucoma gene therapy.

Aged↗
PubMed2004

Role of lysophospholipid growth factors in the modulation of aqueous humor outflow facility.

PURPOSE: To investigate the role of lysophospholipid growth factors in the regulation of aqueous humor outflow in the trabecular meshwork (TM). METHODS: The expression profile of the endothelial differentiation gene (Edg) family of G-protein coupled receptors was determined by RT-PCR of human TM (HTM) cell-derived total RNA and by PCR amplification of HTM cell-derived and tissue-derived cDNA libraries. The effects of lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) on actin cytoskeleton and focal adhesions and on myosin light-chain (MLC) phosphorylation in HTM cells were evaluated by immunofluorescence microscopy and Western blot analysis, respectively. Activation of Rho GTPase in HTM cells was quantified by "pull-down" assays. Mobilization of intracellular calcium in HTM cells was determined using spectrofluorometric digital-imaging microscopy. The effects of LPA and S1P on aqueous humor outflow facility were evaluated by perfusion of enucleated porcine eyes. RESULTS: Each of the receptor isoforms Edg1, -2, -3, and -4 was readily detectable in three of four HTM cell-derived libraries, whereas Edg2 was detectable in the HTM tissue library. LPA (20 microM) and S1P (1 microM) stimulated actin stress fiber and focal adhesion formation, increased MLC phosphorylation, and induced marked activation of Rho GTPase in HTM cells. Both LPA (20 microM) and S1P (10 microM) also stimulated increases in intracellular calcium concentration in HTM cells. LPA- and S1P-induced effects on MLC phosphorylation in HTM cells were markedly inhibited by pretreatment with the Rho kinase-specific inhibitor Y-27632 (5 microM). Perfusion of LPA (50 microM) and S1P (5 microM) in enucleated porcine eyes produced a significant decrease in aqueous humor outflow facility from baseline of 37% (n = 6) and 31% (n = 5), respectively. CONCLUSIONS: These studies demonstrate that LPA and S1P, the physiological agonists of Edg receptors, decrease outflow facility in perfused porcine eyes in association with increased MLC phosphorylation and Rho guanosine triphosphatase (GTPase) activation. These data provide evidence for a novel mechanism for negative regulation of outflow facility, which may contribute to overall physiological homeostasis of aqueous humor outflow facility.

Actins↗
PubMed2006

Matrix metalloproteinases and their inhibitors in the chamber angle of normal eyes and patients with primary open-angle glaucoma and exfoliation glaucoma.

BACKGROUND: In glaucoma, extensive pathological changes occur in the trabecular meshwork (TM) and juxtacanalicular tissue of the chamber angle. Aqueous humor drainage is disturbed due to the accumulation of extracellular matrix (ECM) material in the outflow system. Matrix metalloproteinases (MMPs) remodel ECM material and, thus, they may have a role in regulating outflow facility and intraocular pressure (IOP). This study examined the expression of MMPs and tissue inhibitors of MMPs (TIMPs) in the chamber angle of normal eyes and in primary open-angle glaucoma (POAG) and in exfoliation glaucoma (ExG). METHODS: TM tissues were isolated from healthy donor eyes for corneal transplantation. Specimens of the inner wall of Schlemm's canal and the juxtacanalicular tissue were collected from patients with POAG or ExG during deep sclerectomy operation. Monoclonal antibodies against MMPs (MMP-1, -2, -3, and -9) and antibodies against TIMPs (TIMP-1, -2, and -3) were used for immunohistochemical staining. RESULTS: Immunoreactivity for MMP-2, TIMP-2, or TIMP-3 was observed in human normal TM and in the inner wall of Schlemm's canal. In general, immunoreactions for all of the tested MMPs were more intense in POAG samples than in ExG samples or in the control group. The only exception was the MMP-2 level, which was the highest in the control group. The staining intensity of MMP-1 or MMP-3 was significantly higher in POAG when compared to ExG. TIMP-1 was significantly increased in POAG compared with ExG and there were no marked differences in the levels of TIMP-2 or TIMP-3 between POAG and ExG. The ratios of MMP-1/TIMP-1 and MMP(1+2+3+9) and TIMP(1+2+3) were significantly higher in samples from POAG compared to those of ExG. CONCLUSIONS: Our results reveal an expression imbalance between MMPs and their endogenous tissue inhibitors in tissue samples from patients with POAG and ExG. Differences in immunohistochemical reactions reflect discrete local pathogenic mechanisms involved in POAG and ExG. With respect to the proposed role of MMPs in the remodeling of ECM material, this may point to a weaker reactivity to the accumulation of ECM material in TM in ExG than POAG eyes.

Aged↗
PubMed2000

Age-dependent prevalence of mutations at the GLC1A locus in primary open-angle glaucoma.

PURPOSE: To screen a population with primary open-angle glaucoma for mutations in the gene that encodes the trabecular meshwork inducible glucocorticoid response protein (TIGR), also known as myocilin (MYOC). METHODS: Ophthalmologic information was collected for study subjects with primary open-angle glaucoma and their relatives. Mutation screening of 74 primary open-angle glaucoma probands was conducted by sequencing TIGR/MYOC coding sequence and splice sites. RESULTS: In 23 families we detected 13 nonsynonymous sequence changes, nine of which appear to be mutations likely to cause or contribute to primary open-angle glaucoma. Two mutations, Arg272Gly and Ile499Ser, and one nonsynonymous sequence variant, Asn57Asp, are novel. We found mutations in nine of 25 juvenile glaucoma probands (36%) and two of 49 adult-onset glaucoma probands (4%). Age classification of families rather than individual probands revealed mutations in three of nine families with strictly juvenile primary open-angle glaucoma (33%), and no mutations in 39 families with strictly adult-onset primary open-angle glaucoma (0%). In families with mixed-onset primary open-angle glaucoma containing both juvenile primary open-angle glaucoma and adult-onset primary open-angle glaucoma cases, we found mutations in eight of 26 families (31%). CONCLUSIONS: Our data suggest that Gly252Arg, Arg272Gly, Glu323Lys, Gln368STOP, Pro370Leu, Thr377Met, Val426Phe, Ile477Asn, and Ile499Ser are likely to play roles that cause or contribute to the etiology of autosomal dominant primary open-angle glaucoma. Our finding of more TIGR/MYOC mutations in families with mixed-onset primary open-angle glaucoma than in the families with strictly adult-onset primary open-angle glaucoma implies that the presence of relatives with juvenile primary open-angle glaucoma in a family could be used as a basis for identifying a subset of the population with adult-onset primary open-angle glaucoma with higher prevalence of TIGR/MYOC mutations. To address this issue, and to refine estimations of mutation prevalence in these age-defined subpopulations, prospective study of a larger population ascertained entirely through adult-onset primary open-angle glaucoma probands will be needed.

Adolescent↗
PubMed2006

Ultrastructural and biochemical evaluation of the porcine anterior chamber perfusion model.

PURPOSE: To evaluate a porcine anterior chamber perfusion model and to test the transferability of data obtained with this model to the human system. METHODS: Porcine eyes were obtained from a local abattoir and processed within 2 hours after death. Anterior chambers of 42 pairs of eyes were dissected with removal of lens, vitreous, iris, and ciliary processes and perfused for 72 (40 pairs) or 140 (2 pairs) hours with medium or medium supplemented with 10 ng/mL transforming growth factor (TGF)-beta2. Facility was continuously measured. Afterward, trabecular meshwork (TM) specimens from all quadrants were prepared, and sections were analyzed morphologically and with immunohistochemical methods. TM sections of 10 nonperfused pairs of eyes were used as the control. RNA and protein was extracted from the TM specimens. Expression of alphaB-crystallin, fibronectin (FN), plasminogen activator inhibitor (PAI)-1, thrombospondin (TSP)-1, and connective tissue growth factor (CTGF) mRNA and protein in medium-perfused and TGF-beta2-perfused anterior segments was examined by Northern and Western blot analyses. RESULTS: The nonperfused TM showed prominent differences between the temporal and nasal quadrants. Temporally, the ciliary muscle (CM) was pronounced, the scleral sulcus was long and flat, and the scleral spur extended toward the iris root. Nasally, the CM was thin, the sulcus deep, and the spur compact. The outer TM was expanded between the scleral spur and cornea throughout the entire circumference. On the ultrastructural level, the elastic network was connected to the cribriform TM cells and the aqueous plexus endothelium. Perfusion itself had only small effects on the morphology of the outer TM. Aqueous plexus loops remained open, and TM cells showed no signs of necrosis or pyknosis. alphaB-crystallin expression was significantly increased in perfused eyes. Perfusion with TGF-beta2 for 72 hours reduced outflow facility to approximately 60% of that of the medium-perfused control. TM cells adjacent to putative drainage pathways showed enlarged cisterns of rough endoplasmic reticulum (rER), a sign of active protein synthesis. Expression of alphaB-crystallin and FN mRNA were elevated by factors of 5 and 3, respectively. The proteins were upregulated by a factor of 2.5. In addition, TGF-beta2 upregulated PAI-1 (1.7-fold) and TSP-1 (1.6-fold) proteins, two factors shown to be TGF-beta2 responsive in human TM cell culture experiments. CTGF expression was not altered. CONCLUSIONS: These new ultrastructural investigations indicate that the cribriform and subendothelial regions of the porcine TM have an architecture similar to that of the primate TM. The biochemical and physiological response to TGF-beta2 was identical with that described in human TM cell culture and anterior chamber perfusion. The porcine anterior chamber perfusion model is valid for the human system.

Animals↗
PubMed2006

Caldesmon effects on the actin cytoskeleton and cell adhesion in cultured HTM cells.

Caldesmon is a multifunctional ubiquitous regulator of the actin cytoskeleton, which can affect both actomyosin contractility and actin polymerization. Previous studies showed that caldesmon over-expression in cultured fibroblasts produces effects that resemble those of chemical inhibitors of cellular contractility. Since these inhibitors (H-7, Y-27632, etc.) have been shown to lower intraocular pressure and increase outflow facility from the anterior chamber of the eye, we proposed that caldesmon might be used for gene therapy of glaucoma. In the present study we examined the effects of expression of adenovirus-delivered rat non-muscle caldesmon fused with green fluorescent protein (AdCaldGFP) on the actin cytoskeleton and matrix adhesions in cultured human trabecular meshwork (HTM) cells. In addition, we assessed the effect of caldesmon on the stability of cell-cell junctions in kidney epithelial MDCK cells. Cultured HTM cells demonstrate a well-developed actin cytoskeleton, comprising mainly arrays of parallel actomyosin bundles (stress fibers). Lamellipodial protrusions containing dense actin networks are also observed. Cell-matrix adhesions are dominated by focal adhesions (FAs) associated with the ends of the stress fibers, focal complexes in lamellipodia, and fibrillar adhesions in the central part of the spread cells. Treatment of HTM cells with AdCaldGFP resulted in dose-dependent morphological changes within 24-48 hr post-infection. Cells expressing moderate levels of caldesmon exhibited straight bundles containing actin and myosin II, which were considerably shorter than those in control cells. Short filament bundles in caldesmon over-expressing cells formed arrays consisting of triangular actin structures with small vinculin-positive FAs at their vertices. In addition, the fraction of cells displaying large lamellipodia increased. About 40-50% of the population of caldesmon-expressing cells demonstrated high levels of GFP-caldesmon expression and severe changes in the actin cytoskeleton, manifested by the disappearance of stress fibers and the formation of curved actin- and myosin-containing bundles. These bundles formed together a dynamic network consisting of pulsating loops filling the entire cytoplasm. Addition of thapsigargin, which increases intracellular Ca++ concentration, resulted in a straightening of the curved bundles. Another type of novel actin structures induced by caldesmon over-expression were highly dynamic circular waves that propagated over the affected cells with a velocity about 10 microm min. In cells with disrupted stress fibers, vinculin-containing FAs and tensin-rich fibrillar adhesions had also essentially vanished. However, phosphotyrosine-positive focal complexes were still prominent throughout the lamellipodia of these cells. Over-expression of caldesmon in MDCK cells reduced, in a dose dependent manner, the beta-catenin content at cell-cell adherens junctions and in some cases led to physical disruption of adherens junctions. Thus, caldesmon over-expression induces unique reorganization of the actin cytoskeleton in affected cells, accompanied by disruption of focal and fibrillar cell-matrix adhesions, and destabilization of cell-cell adherens junctions. Inducing such changes in the contractility and actin cytoskeleton of HTM cells in glaucomatous eyes in vivo could produce a therapeutically useful increase in outflow facility.

Actins↗
PubMed2004

Experimental and numerical studies of adenovirus delivery to outflow tissues of perfused human anterior segments.

PURPOSE: To investigate the efficacy of two different methods of adenoviral transfer of genes to trabecular meshwork (TM) and Schlemm's canal (SC) cells in cultured human anterior segments, using both experimental and numerical analyses. METHODS: Replication-deficient adenoviruses having coding sequence for beta-galactosidase (beta-gal) under the control of the cytomegalovirus promoter were used. Efficiency of gene transfer over time was verified by infecting cultured human TM cells and assaying for beta-gal activity. Next, ostensibly normal paired human eyes were prepared by standard techniques and perfused for 2 to 5 days to measure baseline facilities. Eyes were then infected by one of two methods: standard transcorneal puncture, or injection into a 1 mm diameter silastic segment of supply tubing immediately upstream of the perfusion dish. In both cases, the nominal total dose was 2 x 10(8) viral particles. Five days after viral injection, eyes were harvested and fixed, and wedges from each of four quadrants were examined histologically. Sections were assayed for beta-gal activity and/or stained with toluidine blue. In a parallel study, flow and viral transport within perfused anterior segments were numerically simulated for conditions that approximated those used experimentally. RESULTS: Eyes receiving viral particles by transcorneal injection showed variable levels of beta-gal activity and highly variable TM cellular morphology, ranging from excellent preservation to cellular lysis. Eyes receiving an equivalent viral dose via the supply tubing showed higher transfer efficiency, as judged by almost complete TM cell loss (indicative of viral toxicity) and intense extracellular beta-gal activity from the residual cytoplasm. At lower doses (1/3 to 1/1000 of that used in transcorneal injection) beta-gal activity was still present, while TM cell morphology was good at the lower viral doses. Computer modeling showed that the region beneath the cornea was nearly stagnant, and consequently virus introduced into this region by transcorneal injection was delivered very slowly to the TM. This caused the effective delivered viral dose to be low and sensitively dependent on the volume and shape of the transcorneally injected virus bolus. CONCLUSIONS: Injection of adenovirus into supply tubing led to consistent delivery of reporter gene and approximately 300-fold greater efficiency of gene transfer compared to the transcorneal injection method, and is therefore the preferred method for introducing viral particles into perfused anterior segments. These findings were consistent with computer modeling of flow and mass transport in perfused anterior segments. Although these quantitative results are specific to adenovirus, this general trend should hold for a wide range of perfused compounds.

Adenoviruses, Human↗
PubMed2003

Signaling pathways used in trabecular matrix metalloproteinase response to mechanical stretch.

PURPOSE: Trabecular meshwork (TM) matrix metalloproteinase (MMP), and tissue inhibitor (TIMP) changes in response to mechanical stretching appear to be central to intraocular pressure (IOP) homeostasis. Studies were conducted to define the signal transduction pathway responsible for the increases in MMP-2 and -14 that occur in response to mechanical stretching of TM cells. METHODS: Porcine TM cells were subjected to mechanical stretching, and changes in MMP-2 and -14 levels were determined by gelatin zymography and Western immunoblot analysis. Effects of signal transduction pathway inhibitors on MMP levels were analyzed. Phosphospecific antibodies were used to identify phosphorylation changes in select pathway intermediates. In silico secondary structure analysis was conducted on the 5' untranslated regions (UTRs) of MMP-2 and -14 mRNAs. RESULTS: The increases in MMP-2 and -14 that occur 24 hours after sustained mechanical stretching of TM cells were blocked by rapamycin. Wortmannin blocked the MMP-2 but not the MMP-14 increase. Protein kinase B (PKB) phosphorylation on S473 and T308 was increased significantly by stretching. Rapamycin-sensitive phosphorylation of T389 in p70/p85 S6 kinase was also increased. The phosphorylations of the translation initiation factor eIF-4E on S209 and of its inhibitory binding protein 4E-BP1 on T70 were both increased by stretch. The calculated free energies of secondary structures of the 5' UTRs of the mRNAs for MMP-2 and -14 were negative and relatively large. MMP-2 also had pyrimidine tracts in the extreme 5' region of its UTR. CONCLUSIONS: The increases in TM MMP-2 and -14 protein levels in response to mechanical stretching appear to be transduced at least in part by mTOR, the mammalian target of rapamycin (mTOR). The wortmannin sensitivity implicates phosphoinositide 3-kinase as a modulator of the MMP-2 but not the MMP-14 increase. Integrin-linked kinase (ILK), phosphoinositide-dependent kinase (PDK-1), and PKB are implicated in the MMP-2 increase. Translational initiation involving eIF-4E and its inhibitory binding protein 4E-BP1 appear to be involved in both the MMP-2 and -14 increases with stretching and are normally regulated by mTOR. The high degree of secondary structure in the 5' UTRs of these transcripts is typically an indicator of genes specifically sensitive to regulation through this pathway. P70/p85 S6 kinase is probably involved downstream from mTOR and PKB in regulating translation of MMP-2, which has pyrimidine tracts in its 5' UTR. Manipulation of these transduction pathways may provide new approaches to therapeutic IOP regulation.

5' Untranslated Regions↗
PubMed2000

Ultrasound biomicroscopy of the anterior segment after congenital cataract surgery.

PURPOSE: To evaluate the structure of the anterior segment after congenital cataract surgery with ultrasound biomicroscopy. METHODS: Ultrasound biomicroscopy was performed on 28 eyes of 15 patients after congenital cataract surgery (age range, 8-27 years; mean, 13.1 +/- 1.1 years), of which 26 eyes had pars plana lensectomy and two eyes had phacoemulsification and aspiration, and on 21 eyes of 11 phakic control subjects who were healthy (age range, 6-17 years; mean, 11.5 +/- 1.1 years). The angle-opening distance at 500 microm was measured on a line perpendicular to the trabecular meshwork 500 microm from the scleral spur, and the trabecular-iris angle was measured. RESULTS: Angle-opening distance at 500 microm in the congenital cataract group ranged from 174 to 920 microm (mean, 424 +/- 28 microm), and in the control group it ranged from 260 to 1,119 microm (mean, 643 +/- 57 microm), a difference that was significantly different (P =.0004, Student t test). The trabecular-iris angle in the congenital cataract group ranged from 30.4 to 66.9 degrees (mean, 41.9 +/- 1.6 degrees) and in the control group ranged from 32.1 to 69.8 degrees (mean, 44.9 +/- 2.5 degrees), a difference that was not significantly different (P =.28, Student t test). This means that there is a tendency for high insertion of the iris in the congenital cataract group, and intraocular pressure was elevated after surgery (including glaucoma and ocular hypertension) in 10 (47.6%) of 21 eyes with high insertion. The pars plicata after congenital cataract surgery was significantly flatter than that of control subjects (P =.0057, Student t test). CONCLUSIONS: Ultrasound biomicroscopy is a useful method for evaluating the anterior segment of eyes with congenital cataract, and complications after cataract surgery can be caused by disorganization of the anterior segment. Patients with abnormalities of the anterior segment should be followed up closely for the development of glaucoma.

Adolescent↗
PubMed1999

Corticosteroids and glaucoma risk.

Corticosteroids (glucocorticoids), used frequently as potent anti-inflammatory agents, increase the risk of glaucoma by raising the intraocular pressure (IOP) when administered exogenously (topically, periocularly or systemically) and in certain conditions of increased endogenous production (e.g. Cushing's syndrome). Approximately 18 to 36% of the general population are corticosteroid responders. This response is increased to 46 to 92% in patients with primary open-angle glaucoma (POAG). Patients over 40 years of age and with certain systemic diseases (e.g. diabetes mellitus, high myopia) as well as relatives of patients with POAG are more vulnerable to corticosteroid-induced glaucoma. The association of corticosteroid-induced ocular hypertension in other conditions which are considered as risk factors for glaucoma (racial origins, hypertension, migraine, vasospasm) is likely but not fully established. The proposed mechanism of corticosteroid-induced glaucoma includes morphological and functional changes in the trabecular meshwork system and is similar to the pathogenesis of POAG. Trabecular cells exposed to corticosteroids in vitro show endoreplication of nuclei, an increase in cell size and excessive production of an approximately 56kD glycoprotein, identified as myocilin and transcribed by the GLC1A gene. Induction of ocular hypertension after corticosteroid administration depends on the specific drug, the dose, the frequency of administration and the corticosteroid responsiveness of the patient. The risk of corticosteroid-induced glaucoma can be minimised with judicious use of corticosteroids, as well as education of patients and medical practitioners. New treatment modalities include modified steroids and nonsteroidal anti-inflammatory agents that will have less effect on the elevation of IOP.

Glaucoma↗
PubMed1994

Trigeminal innervation of the scleral spur in cynomolgus monkeys.

Degeneration induced by intracranial section of the ophthalmic branch of the trigeminal nerve was used as a marker to trace the ocular passage and termination of fibres in ocular filtration angle structures of 3 cynomolgus monkeys. Fine supraciliary nerves branched from precorneal nerves as they entered the sclera from the uvea, passed forwards radially in the lamina fusca, entered the scleral spur, turned circumferentially and terminated. Many of the fibres were degenerated. Other less numerous branches advanced in the ciliary muscle close to the lamina fusca and were distributed either to the spur or to the trabecular meshwork. Spur fibres were approximately 4 times as numerous as trabecular fibres. The position, arrangement and ophthalmic identity of most of the spur terminals suggest a capacity to record tension produced by ciliary muscle contraction.

Animals↗
PubMed1994

Hyaluronan in the bovine ocular anterior segment, with emphasis on the outflow pathways.

PURPOSE: It has been postulated that glycosaminoglycans play a role in the regulation of outflow resistance. The purpose of these studies was to localize the distribution of hyaluronan (HA) in the anterior segments of bovine eyes to understand better the possible role of HA in the outflow pathway. METHODS: Eyes from four 2-week-old calves and four 1-year-old cows were examined using a biotinylated-hyaluronan binding protein to localize HA in tissue sections of the anterior segment of bovine eyes. Various fixations and microwave irradiation were compared. The vitreous body in each section served as a positive control. Sections treated with Streptomyces hyaluronidase were used to confirm specificity. RESULTS: No significant difference in distribution of HA was found between various fixations and between calves and cows. HA was found in subconjunctival connective tissue and in intercellular spaces of limbal and conjunctival epithelium, but not in corneal epithelium. Staining was sometimes found on the surface of the corneal epithelium and endothelium, as well as on the conjunctival epithelium. No staining was found within the corneal stroma. There was HA in iris stroma, but not in the root of the iris or ciliary body stroma. HA was present in the anterior, nonfiltering part of the trabecular meshwork (TM) and surrounding collector channels and blood vessels in the sclera; to a lesser extent, it was present in the juxtacanalicular region of the TM. HA was detectable neither within trabecular beams nor filling the intertrabecular spaces. Strong staining was found, however, in the nerve bundles in the angle region. Staining for HA in vitreous was invariably positive, and in Streptomyces hyaluronidase-treated sections it was negative. CONCLUSION: HA was not uniformly distributed in the bovine TM. The distribution of HA in the flow pathway of the aqueous outflow system indicates that only a small fraction of the HA found in biochemical analyses of the bovine meshwork is located in the region where the flow resistance is thought to reside.

Animals↗
PubMed1975

[Transtrabecular iridectomy - a new surgical technique in the treatment of chronic narrow-angle glaucoma (author's transl)].

A new surgical technique in the treatment of chronic narrow-angle glaucoma is described. After forming a scleral flap and opening of Schlemm's canal a probe is inserted into the canal. We dissect the sclera and the outer wall of Schlemm's canal by an oblique cut. The probe being retracted again, the trabecular meshwork is cut and a basal iridectomy is performed. Then the scleral cut and the trabecular window are closed tightly. Postoperative pressure behaviour and gonioscopy indicate, that the outflow of aqueous humour occurs via trabecular cleft and the collector channels in the operating field; obviously with this technique the intrascleral outflow channels can be saved to a great extend.

Chronic Disease↗
PubMed2003

Why is glaucoma associated with exfoliation syndrome?

Exfoliation syndrome (XFS) is an age-related, generalized disorder of the extracellular matrix characterized by production and progressive accumulation of a fibrillar material in tissues throughout the anterior segment and also in connective tissue portions of various visceral organs. Mature exfoliation fibrils are composed of 8-10 nm microfibrils resembling elastic microfibrils. The exact chemical composition of exfoliation material (XFM) remains unknown. It appears to consist of a complex glycoprotein/ proteoglycan structure composed of a protein core surrounded by abundant glycoconjugates. The protein components include both non-collagenous basement membrane components and epitopes of the elastic fiber system, particularly components of elastic microfibrils. Overall, XFS is the most common identifiable cause of glaucoma, accounting for the majority of cases in some countries, and causing both open-angle glaucoma and angle-closure glaucoma. Iridolenticular friction leads to loss of XFM from the anterior lens surface and disruption of the iris pigment epithelium, resulting in pigment deposition in the trabecular meshwork, which also produces XFM locally. The primary cause of chronic pressure elevation appears to be the active involvement of trabecular cells and Schlemm's canal cells in particular, in the generalized pathologic matrix process with subsequent degenerative changes of Schlemm's canal and adjacent tissues. Narrow angles and angle-closure are common in XFS. Pupillary block may be caused by a combination of posterior synechiae, increased iris thickness or rigidity, or anterior lens movement secondary to zonular weakness or dialysis. Enlargement of the lens due to cataract formation and relative pupillary constriction are additional factors.

Animals↗
PubMed1990

Effects and interactions of epinephrine, norepinephrine, timolol, and betaxolol on outflow facility in the cynomolgus monkey.

Total outflow facility was determined in cynomolgus monkeys by two-level constant pressure perfusion. Topical epinephrine increased facility by 30% to 35% three to four hours after dosing, whether given as a single 600-micrograms dose or as twice daily 600-micrograms doses for three days. A single 5.5-micrograms intracameral dose of epinephrine or norepinephrine increased facility by 65% to 70% three to four hours after dosing. A single 180-micrograms topical dose of timolol or betaxolol had no effect on facility three to four hours later. Timolol pretreatment prevented the facility-increasing effect of both topical epinephrine and intracameral norepinephrine, but betaxolol pretreatment prevented neither. These findings indicate that no cumulative facility-increasing effect of epinephrine, beyond the acute (three hour) facility-increasing effect, develops within three days; there may be a facility-decreasing effect of large topical epinephrine doses on the vascular structures external to the trabecular meshwork; there is no, or only subthreshold, facility-affecting ambient beta-adrenergic tone in the meshwork; and the facility-increasing effect of both epinephrine and norepinephrine is mediated by beta 2-adrenergic receptors in the trabecular endothelium.

Administration, Topical↗