Endothelial Tyrosine Kinase Tie1 Is Required for Normal Schlemm's Canal Development-Brief Report.

BACKGROUND: Schlemm's canal (SC) is a large vessel residing in the iridocorneal angle and is required to regulate aqueous humor outflow. Normal SC structure and function is indispensable for maintaining normal intraocular pressure, and elevated intraocular pressure is a risk factor for development of glaucoma. Recent reports have identified a key role of the angiopoietin-Tie2 pathway for SC development and function; however, the role of the orphan receptor Tie1 has not been clarified. METHODS: We used Tie1 knock out mice to study the function of Tie1 in SC development and function. Real-time quantitative polymerase chain reaction and Western blot analyses were used to verify Tie1 deletion. High-resolution microscopy of mouse SC whole mount and cross sections were used to study SC morphology. Measurement of intraocular pressure in live mice was used to study the impact of Tie1 on SC function. RESULTS: Tie1 is highly expressed in both human and mouse SC. Tie1 knock out mice display hypomorphic SC and elevated intraocular pressure as a result of attenuated SC development. CONCLUSIONS: Tie1 is indispensable for SC development and function, supporting it as a novel target for future SC-targeted glaucoma therapies and a candidate gene for glaucoma in humans.

Augmentation of Lysosomal Phospholipase A2 Activity in the Anterior Chamber in Glaucoma.

AIM: The lysosomal enzyme in the anterior chamber has a crucial role in the digestion of the insoluble materials in the aqueous humor (AH). The dysfunction of AH filtration in the trabecular meshwork (TM) causes increasing AH outflow resistance in the TM. Those insoluble objects, including phospholipids, should be digested in the TM for normal outflow. The present study was conducted to explore the involvement of lysosomal phospholipase A2 (LPLA2), a phospholipid-degrading enzyme, of the AH in glaucoma using clinical AH specimens. MATERIALS AND METHODS: One hundred and twenty-five AH specimens were collected from patients. The measurement of LPLA2 activity in the AH was carried out using liposomes consisting of phosphatidylglycerol and N-acetylsphingosine (NAS). The correlation between the LPLA2 activity in the AH and ocular diseases was investigated. RESULTS: The human AH showed both transacylation of NAS and the release of fatty acids under acidic conditions but not at a neutral pH, which is consistent with the known properties of LPLA2. The LPLA2 activity in the AH was not affected by age or systemic disease. A comparison between ocular diseases showed that the AH specimens obtained from patients with glaucoma had significantly higher LPLA2 activity than the other ocular disease groups. DISCUSSION: The present findings suggest that the ascended level of LPLA2 activity in the AH of glaucoma patients is associated with the development of glaucoma.

Phospholipase A2 in chamber angle of normal eyes and patients with primary open angle glaucoma and exfoliation glaucoma.

PURPOSE: Phospholipase A2 (PLA2) is a growing family of lipolytic enzymes that play a key role in various biological processes including general lipid metabolism, membrane homeostasis, and in diseases such as atherosclerosis, arthritis, and acute pancreatitis. Oxidative stress as well as inflammation may be associated with glaucoma pathogenesis. Therefore, our aim was to examine the expression of group IIA secretory PLA2 (sPLA2-IIA), group V secretory PLA2 (sPLA2-V), calcium-independent PLA2 (iPLA2), and cytosolic PLA2 (cPLA2) type in the trabecular meshwork (TM) and the canal of Schlemm in normal eyes and in juxtacanalicular tissue samples from patients with primary open angle glaucoma (POAG) or exfoliation glaucoma (ExG). METHODS: TM tissues were isolated from healthy donor eyes for corneal transplantation. Specimens of inner wall of the Schlemm's canal and the juxtacanalicular tissue were collected during deep sclerectomy from the eyes of patients who had POAG or ExG. Antibodies against PLA2s (sPLA2-IIA, sPLA2-V, iPLA2, and cPLA2) and a standard immunohistochemical procedure were used for the analysis. Quantification of immunoreactions was provided using a Photoshop-based image analysis. Double-staining immunofluorescence of macrophages and sPLA2-IIA was performed by using confocal microscopy. RESULTS: sPLA2-IIA was not present in normal TM. In contrast, sPLA2-IIA levels were significantly higher in glaucoma patients than in controls. Furthermore, sPLA2-IIA expression was much higher in POAG when compared to ExG. iPLA2 was found to predominate in normal human TM, and it demonstrated strong labeling in the uveal and corneoscleral meshwork. The staining of juxtacanalicular meshwork was only moderate in density. In contrast, expression of the enzyme was significantly decreased in glaucoma patients, especially in ExG, when compared to normal controls or to POAG. In addition, strong regional differences were detected in sPLA2-IIA and iPLA2 levels in POAG, whereas immunostaining of these enzymes was much lower and rather uniform throughout ExG sample. In POAG, sPLA2-IIA staining was restricted to certain parts of the trabecular samples where sPLA2-IIA positive macrophages were also present. Immunostaining of sPLA2-V or cPLA2 was low, and no significant changes were found in levels of these enzymes between normal and glaucomatous samples. CONCLUSIONS: sPLA2-IIA, an oxidative stress marker in atherosclerosis, is overexpressed especially in POAG. This result supports the hypothesis that oxidative stress may play a significant role in the pathogenesis of POAG. In ExG, a dramatic decrease in the expression level of iPLA2, a housekeeping enzyme in phospholipid remodeling, may indicate imbalance in phospholipid turnover and also inhibition of normal physiological functions in the TM. These findings may contribute to understanding the pathogenesis of POAG and ExG and may be important for the development of novel therapeutic strategies to different glaucomas.

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.

Gene transfer to trabecular meshwork endothelium via direct injection into the Schlemm canal and in vivo toxicity study.

PURPOSE: Our aim was to investigate the efficiency of adenoviral gene transfer via direct injection into the Schlemm canal ex vivo in human donor eyes and to examine the effect of human MMP-3 transgene expression in a rat model in vivo. METHODS: A viscocanalostomy-like operation was performed and adenoviral vector encoding for MMP-3 and green fluorescent protein was injected into human Schlemm canal or rat anterior chamber. RESULTS: Transgene expression was high in trabecular meshwork endothelium in human donor eyes. In vivo, adenovirus caused dose-dependent inflammation. CONCLUSIONS: Direct injection of adenoviral vectors into the Schlemm canal has potential in glaucoma treatment.

Effect of IOP elevation on matrix metalloproteinase-2 in rabbit anterior chamber.

To investigate changes in the level of matrix metalloproteinase-2 (MMP-2) in the anterior chamber of rabbit with intraocular pressure (IOP) elevation. The IOP was elevated with scleral encircling in 12 rabbits. In the control group (4 rabbits), IOP was not changed after scleral encircling, and in group 1 (4 rabbits) and 2 (4 rabbits), IOP was elevated about 10 and 20 mmHg respectively after scleral encircling. At 2 days after scleral encircling, aqueous sampling was performed and levels of MMP-2 were checked by Western blots and gelatin zymograms. The greater the IOP elevation, the more MMP-2 expression in the anterior chamber by Western blots and gelatin zymograms. The increase in MMP-2 expression in response to IOP elevation may have important implications for the IOP feedback control mechanism.

Superoxide dismutase in the anterior chamber and the vitreous of diabetic patients.

Total superoxide dismutase (SOD) activity was examined in the anterior humor of 32 diabetic patients and 34 nondiabetic controls during cataract extraction. Median age (95% confidence interval) was 77.5 yr (73.3-81.0) and 79.3 yr (76.0-83.2), respectively. The SOD activity also was examined in posterior vitreous sampled peroperatively in 10 diabetics with proliferative retinopathy and post-mortem in seven diabetic patients and 35 nondiabetic controls. Ages were 57.2 yr (35.0-73.9), 74.4 yr (40.7-83.6), and 73.8 yr (65.0-80.2), respectively. In nondiabetic patients, the total SOD activity was much lower in the anterior chamber, 9.9 U/ml (8.1-12.6), than in the posterior vitreous, 106.3 U/ml (range 65.6-119.0), P < 0.001. We found no difference between the SOD levels in the anterior chamber of nondiabetic controls and diabetic patients, who had 9.6 U/ml (7.6-13.7). The SOD activity in posterior vitreous in diabetic patients sampled peroperatively, 23.9 U/ml (8.9-39.2), P < 0.0001, and post-mortem, 39.5 U/ml (6.5-214.2), P < 0.04, was significantly lower than in the controls sampled post-mortem, 106.3 U/ml (65.6-119.0). Low levels of SOD in the anterior chamber may be involved in cataract development, in diabetic patients and nondiabetic controls. That diabetics had decreased SOD activity in the posterior vitreous points to a possible role of SOD in the complex process of diabetic retinopathy development.

[The proteinase and alpha 1-antitrypsin activities in the tissues during emotional stress in rabbits]. / Aktivnost' proteinaz i al'fa 1-antitripsina v tkaniakh pri émotsional'nom stresse u krolikov.

The experiments on rabbits under acute emotional stress have revealed a rise in activity of proteinases of blood serum, salivary glands and no changes of this activity in watery moisture of an eye. The activity of alpha-antitrypsin in watery moisture of an eye, unlike the blood serum, significantly increased, that evidently prevented the activation of proteolysis in it. The emotional stress is also accompanied by the intensification of fibrinolysis, one of the most important proteolytic systems of the blood.

Dopamine-beta-hydroxylase-positive nerves in normal and transplanted pancreatic tissue in the anterior eye-chamber of rats.

Dopamine-beta-hydroxylase(DBH)-positive nerves were demonstrated in normal and in pancreatic tissue fragments transplanted for 22 and 32 days into the anterior eye-chamber of rats using immunohistochemical techniques. Dopamine-beta-hydroxylase-immunopositive neurons of different shapes could be observed in normal pancreas. The neurons had either spindle or oval shapes. In the transplanted tissue, DBH-positive neuronal profiles were found in the stroma. In some cases DBH-immunopositive cells appeared as a cluster of cells around pancreatic ducts and blood vessels or as solitary cells. The wall of pancreatic ducts in the transplants also contained DBH-immunopositive nerve profiles.

Hydrogen peroxide in the rabbit anterior chamber: effects on glutathione, and catalase effects on peroxide kinetics.

Intracameral hydrogen peroxide (H2O2) is cleared at a faster rate in young (t1/2, 93 seconds) than in adult (t1/2, 109 seconds) rabbits. Extrapolated zero time concentrations of H2O2 were 3.3 mM in adults and 3.2 mM in young. The more rapid disappearance of H2O2 correlated with greater catalase levels in iris (35%) and corneal endothelium (50%) in young as compared to adult animals. Catalase levels have been found to be reduced in ocular tissues with 3-amino-1H-1,2,4-triazole (3AT) in a dose-related manner up to 6 ml/kg of an intravenous 3M solution. Iris and ciliary processes showed a linear reduction with dose, while corneal endothelium, liver and lung reached near maximal decreases in catalase activity at 2, 4, and 6 ml/kg, respectively. 3AT caused a significant dose-dependent extension of the rate of clearance of H2O2 from the anterior chamber, that was directly related to catalase loss. The t1/2 for H2O2 disappearance in adult animals increased from 109 seconds with no 3AT, to 147 seconds after 2 ml/kg 3M 3AT, to 161 seconds after 4 ml/kg 3M 3AT and 184 seconds after 6 ml/kg 3M 3AT. Corneal endothelial oxidized glutathione levels were transiently increased after intracameral hydrogen peroxide. Considering the sum total of all tissues of the anterior segment, specific incremental decreases of catalase generated by intravenous 3AT caused the t1/2 of H2O2 clearance from the anterior chamber to become longer, while the reducing power of anterior segment tissues excluding lens epithelium is related clearly to the systemic dose of 3AT.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutathione reductase of calf trabecular meshwork.

Hydrogen peroxide has been found in both calf and human aqueous humor at a level of 25 microM. It is likely, therefore, that trabecular meshwork possesses mechanisms for detoxifying H2O2, both to protect itself and other more distal structures of the outflow pathway from oxidative damage. We have recently demonstrated an active glutathione peroxidase in calf trabecular meshwork. In this study, we have characterized the complementary enzyme, glutathione reductase. The activity was present at a level of 0.120 units/min/g wet of tissue (0.005 units/min/mg soluble protein). The enzyme quickly lost activity in crude extracts but could be stabilized by heating at 60 degrees C for 30 min. Denatured protein was removed by centrifuging at 43,000 X g. Heating at 80 degrees C for 10 min destroyed all enzyme activity. Addition of 1 mM GSSG protected the enzyme completely from heat denaturation; NADP+ and GSH offered some protection but NADPH provided none. The supernatant from the 60 degrees C heat treatment was further purified by affinity chromatography on 2',5'-ADP-agarose. Overall purification was 200-fold with a yield of 80%. The pH optimum of the purified enzyme was 7.0. The KmS for NADPH and GSSG were 19 microM and 78 microM, respectively. The heat inactivation properties of the purified enzyme were identical to those in the crude extract. An enzyme activity stain on disc gel electrophoresis showed that the enzyme exists in only one form.

[Redifferentiation and a karyotypic structural change in populations of tumorous rhabdomyoblasts proliferating in the anterior chamber of the eye]. / Redifferentsirovka i izmenenie kariotipicheskoi struktury populiatsii opukholevykh rabdomioblastov pri ikh proiferatsii v perednei kamere glaza.

A study was made of the transplantable rhabdomyosarcoma of mice M-62 during its transplantation after 15 passages simultaneously into the subcutaneous connective tissue and the eye anterior chamber (EAC), the latter being an immunologically advantageous site. The transplantable rhabdomyosarcoma cell populations, proliferating in EAC, are characterized by a higher differentiation level as compared with those proliferating in the subcutaneous connective tissue, the cells being taken from the same tumour, at the same passage. Morphologically it is expressed in the appearance of cytotypical (myofibrils in mononuclear tumour myoblasts) and histotypical (formation of myosymplasts) characters of differentiation. The increase in the intensity of differentiation correlates with changes in the ratios of M- and H-forms of LDH towards that which is characteristic of the definitive skeletal muscle tissue. A more intensive differentiation occurs on the background of changes in the karyotype structure of the populations of tumour myoblasts expressed in the increased share of diploid cells. An assumptions is made that the populations of tumour cells proliferating in EAC, i. e. under conditions of isolation from the immunological reactions of the organism, demonstrate changes towards normalization.

[Changes in the LDH isoenzyme spectrum in transplantable rhabdomyosarcoma clones transplanted into the anterior chamber of the eye]. / Izmenenie spektra izozimov LDG v klonakh perevivnykh rabdomiosarkom, transplantirovannykh v peridniuiu kameru glaza.

Results of gel electrophoresis of transplantable rat (RA-2) and murine (MC-53) rhabdomyosarcomas show that the LDH isoenzyme spectrum of tumors growing subcutaneously differ from that of normal muscle tissue, LDH-1 and LDH-2 isoenzyme being absent in zymograms. Using the lung colony formation technique, clones of RA-2 and MC-53 were obtained and their zymograms were investigated. Clone populations of both the tumors were polymorphic for their LDH spectrum, in some clones of RA-2 normal LDH isoenzyme spectrum was found. After a 16-18 day cultivation in rat and mouse eye anterior chamber, LDH spectra of RA-2 and MC-53 clones changed sharply, LDH-1 and LDH-2 appearing. As a result, LDH isoenzyme spectra of some clones that grew in the eye anterior chamber became equal to those of the normal muscle tissue. The observed changes are discussed as biochemical signs of cytodifferentiation of tumor cells.