Adenovirus conducted connective tissue growth factor on extracellular matrix in trabecular meshwork and its role on aqueous humor outflow facility

Deposition of extracellular matrix (ECM) in trabecular meshwork, such as fibronectin, collagen IV, elastin. leads to increased resistance of trabecular meshwork in primary open angle glaucoma (POAG). Connective tissue growth factor (CTGF) is known to regulate the ECM deposits. In this study, we detect the effect of adenovirus conducted CTGF (Adv-CTGF) transfection on either the expression of ECM components or aqueous humor outflow facility. Adv-CTGF was used to transfect rat trabecular meshwork cells in vivo and in vitro. Aqueous humor outflow facility was test by microbeads perfusion. Protein expression of CTGF, fibronectin, and collagen IV was determined using Western blot. In the Adv-CTGF group, the outflow facility displayed a significant decrease from baseline. It appears as though the transfection with Adv-CTGF significantly affects the aqueous humor outflow pattern. A negative correlation between IOP and PEFL indicated that a decrease in the area of bead deposition corresponded to an overall decrease of outflow, leading to an elevated IOP. Adv-CTGF can enhance the expression of CTGF, fibronectin and collagen IV. CTGF is the novel target for treatment of POAG. It is necessary to further study to test inhibition of CTGF expression for treatment of POAG.     

Osteoblasts isolated from mouse calvaria initiate matrix mineralization in culture

A method is presented for isolating osteoblasts from newborn mouse calvaria without the use of digestive enzymes. The procedure is based on the ability of osteoblasts to migrate from bone onto small glass fragments (Jones, S.J., and A. Boyde, 1977, Cell Tissue Res., 184:179- 193). The isolated cells were cultured for up to 14 d in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 50 micrograms/ml of ascorbic acid. 7-d cultures were incubated for 24 h with [3H]proline. High levels of collagen synthesis relative to total protein were found, as measured by collagenase digestion of medium and cell layer proteins. Analysis of pepsin-digested proteins from the same cultures by SDS PAGE showed that type I collagen was predominantly produced with small amounts of type III and V (alpha 1 chains) collagens. Osteoblasts grown in the presence of beta-glycerophosphate were able to initiate mineral deposition in culture. Electron microscopic analysis of the cultures revealed the presence of needle- shaped apatite-like crystals associated with collagen fibrils and vesicles in the extracellular space. Mouse skin fibroblasts cultured under identical conditions failed to initiate mineralization. Electron histochemical studies revealed the presence of alkaline phosphatase activity, associated with osteoblast membranes, matrix vesicles and on or near collagen fibrils. Thus these isolated osteoblasts retained in culture their unique property of initiating mineralization and therefore represent a model of value for studying the mineralization process in vitro.     

Antithrombin III, a serpin family protease inhibitor, is a major heparin binding protein in porcine aqueous humor

Our hypothesis is that the proteins in aqueous humor may be involved in the regulation of outflow facility through the trabecular meshwork and uveoscleral meshwork. In this study, we analyzed the profile of heparin-binding proteins present in porcine aqueous humor to identify and characterize secretory proteins with a binding affinity for heparin. A single step involving heparin-sepharose affinity chromatography of porcine aqueous humor yielded a approximately 60 kDa protein as the major heparin-binding species. This protein was specifically eluted from the column by heparin. The N-terminal sequence and immunological cross reactivity of this protein confirmed its identity as antithrombin III. Aqueous humor from different species, as well as cells from human trabecular meshwork, Schlemm's canal, and lens epithelium, contained detectable amounts of antithrombin III. Based on its known anticoagulative function in endothelial cells and effects on the production of prostacyclin, it is reasonable to speculate that antithrombin III present in aqueous humor might influence the physiology of the trabecular and uveoscleral meshwork and thereby regulate intraocular pressure.     

Trabecular meshwork's collagen network formation is inhibited by non-pigmented ciliary epithelium-derived extracellular vesicles

The present research aims to determine whether the application of non-pigmented ciliary epithelium cells derived extracellular vesicles to human trabecular meshwork cells affects the formation and secretion of collagen type I to the extracellular matrix formation. Following the extraction of non-pigmented ciliary epithelium derived extracellular vesicles by a precipitation method, their size and concentration were determined using tunable resistive pulse sensing technology. Extracellular vesicles were incubated with trabecular meshwork cells for 3 days. Morphological changes of collagen type I in the extracellular matrix of trabecular meshwork cells were visualized using confocal microscopy and scanning electron microscopy. A Sirius Red assay was used to determine the total amount of collagen. Finally, collagen type I expression levels in the extracellular matrix of trabecular meshwork cells were quantified by cell western analysis. We found that non-pigmented ciliary epithelium extracellular vesicles were very effective at preventing collagen fibres formation by the trabecular meshwork cells, and their secretion to the extracellular matrix was significantly reduced (P < .001). Morphological changes in the extracellular matrix of trabecular meshwork cells were observed. Our study indicates that non-pigmented ciliary epithelium extracellular vesicles can be used to control collagen type I fibrillogenesis in trabecular meshwork cells. These fibrils net-like structure is responsible for remodelling the extracellular matrix. Moreover, we suggest that targeting collagen type I fibril assembly may be a viable treatment for primary open-angle glaucoma abnormal matrix deposition of the extracellular matrix.     

Novel molecular insights into RhoA GTPase-induced resistance to aqueous humor outflow through the trabecular meshwork

Impaired drainage of aqueous humor through the trabecular meshwork (TM) culminating in increased intraocular pressure is a major risk factor for glaucoma, a leading cause of blindness worldwide. Regulation of aqueous humor drainage through the TM, however, is poorly understood. The role of RhoA GTPase-mediated actomyosin organization, cell adhesive interactions, and gene expression in regulation of aqueous humor outflow was investigated using adenoviral vector-driven expression of constitutively active mutant of RhoA (RhoAV14). Organ-cultured anterior segments from porcine eyes expressing RhoAV14 exhibited significant reduction of aqueous humor outflow. Cultured TM cells expressing RhoAV14 exhibited a pronounced contractile morphology, increased actin stress fibers, and focal adhesions and increased levels of phosphorylated myosin light chain (MLC), collagen IV, fibronectin, and laminin. cDNA microarray analysis of RNA extracted from RhoAV14-expressing human TM cells revealed a significant increase in the expression of genes encoding extracellular matrix (ECM) proteins, cytokines, integrins, cytoskeletal proteins, and signaling proteins. Conversely, various ECM proteins stimulated robust increases in phosphorylation of MLC, paxillin, and focal adhesion kinase and activated Rho GTPase and actin stress fiber formation in TM cells, indicating a potential regulatory feedback interaction between ECM-induced mechanical strain and Rho GTPase-induced isometric tension in TM cells. Collectively, these data demonstrate that sustained activation of Rho GTPase signaling in the aqueous humor outflow pathway increases resistance to aqueous humor outflow through the trabecular pathway by influencing the actomyosin assembly, cell adhesive interactions, and the expression of ECM proteins and cytokines in TM cells.     

Collagen synthesized and modified by aging fibroblasts in culture.

Collagen is produced by WI-38 diploid human fibroblast cultures throughout their life cycle. It is examined by a sensitive method based on the analysis of specific peptides obtained after digestion with bacterial collagenase. The production and hydroxylation of the collagen is strongly dependent upon the age (population doublings) of the culture and the presence of ascorbic acid. Young cultures (passage 26) produce large amounts of collagen in the absence of ascorbic acid, and this collagen is about 50% hydroxylated compared to that produced by young cultures in the presence of ascorbic acid. Ascorbic acid reduces to about one-half the amount of collagen produced by these young cultures. The young confluent cultures also depend strongly on ascorbic acid for hydroxylation of proline. The dependence declines rapidly with the age of the culture. The collagen produced by young cultures supplied with ascorbic acid is very similar to the type I collagen produced by normal individuals and has about the same degree of hydroxylation of its prolyl residues. The amount of collagen produced by "older" cultures is unaffected by ascorbic acid, but the degree of hydroxylation is normal only if ascorbic acid is present, and is decreased to about 60 to 70% in the absence of the vitamin. "Senescent" cultures showed little, if any, dependency on ascorbic acid, and the collagen produced, with and without the vitamine, is about 80% hydroxylated. The prolyl hydroxylation system of the WI-38 cells and the various controls on the system are age-dependent.     

Measuring triamcinolone acetonide in aqueous humor by gas chromatography-electron-capture negative-ion mass spectrometry

Intravitreal triamcinolone acetonide (IVTA) injection has been used in the treatment of various posterior segment diseases. One of the side effects of IVTA is raised intraocular pressure, which may be secondary to triamcinolone acetonide (TAA)'s effects on the trabecular meshwork that affects aqueous outflow. In order to study the biological effects of TAA on the trabecular meshwork, we firstly need to reliably and accurately detect the concentration of TAA in tissue cells or fluids. In this study we have described a technique of using gas chromatography-electron-capture-negative-ion mass spectrometry (GC-NCI-MS) to develop a simple, sensitive, selective and validated method to detect TAA in aqueous humor (AH) of rabbits following IVTA and subconjunctival TAA injections. We derivatized TAA from extracted aqueous sample by acetic anhydride and BSTFA, respectively, and analyzed by GC-NCI-MS. The detection limit was 0.3ng/ml, linearity over 0.995 from 0 to 300ng/ml. The reproducibility ranged from 10.4 to 3.9 for concentrations from 3 to 300ng/ml, and recovery was over 95% for the concentrations 10, 60, and 200ng/ml. No interference was found from 159 aqueous samples. There was no TAA residue carried to the next injection from previously high concentration injection, 10,000ng/ml. We have provided an alternative, rapid, and robust method other than LC-MS-MS for TAA detection in AH.     

Reduced viability of vascular endothelial cells by high concentration of ascorbic acid in vitreous humor

Normal mammalian vitreous humor maintains its avascularity after regression of hyaloid vessels. Neovascularization in adults is only detected under pathological conditions which suggests that antiangiogenic factors are present in the vitreous humor. To elucidate the mechanism of vitreal angiogenic inhibition, we investigated the effect of vitreous humor on cultured vascular endothelial cells. When bovine aortic endothelial cells were cultured in the presence of bovine vitreous humor in medium, a decrease in cell viability was observed within 24 h. Ascorbic acid from vitreous humor has been identified as a cell death inducing factor with high performance liquid chromatography (HPLC) and molecular mass analysis. Ascorbic acid reduced endothelial cell viability at concentrations normally present in vitreous humor. This effect was completely inhibited by antioxidants, N-acetylcysteine and catalase. Amongst the ascorbic acid derivatives tested, ascorbic acid 2-phosphate did not induce cell death, suggesting that the production of ascorbyl radical is required for induction of cell death. Furthermore, capillary formation in three-dimensional collagen gel cultures characteristic of vascular endothelial cells were disrupted in the presence of ascorbic acid. Since ascorbic acid is highly concentrated in ocular tissues, especially in vitreous humor, it may function as a neovascularization inhibitor.     

Regulation of angiogenesis in vitro by collagen metabolism

Summary The role of collagen in microvascular growth was investigated using the aortic ring model of angiogenesis. Collagen production by vasoformative outgrowths in plasma clot culture of rat aorta was either stimulated with ascorbic acid or inhibited with the proline analogue cis-hydroxyproline. Microvessels proliferating in the absence of ascorbic acid supplements became ectatic and developed large lumina. In contrast, newly formed microvessels in the presence of ascorbic acid remained small and maintained thin lumina throughout the angiogenic process. Biochemical studies demonstrated enhanced collagen production and deposition in cultures treated with ascorbic acid. Ultrastructural studies of these cultures showed a marked increase in newly formed interstitial collagen in the perivascular matrix and in regions of the plasma clot containing nonendothelial mesenchymal cells. Small microvessels with thin lumina similar to the ones observed in ascorbic acid-treated plasma clot cultures were obtained by growing aortic explants in gels of interstitial collagen in the absence of ascorbic acid. Inhibition of collagen production with the proline analogue cis-hydroxyproline had a marked anti-angiogenic effect in both plasma clot and collagen gel cultures. The anti-angiogenic effect of cis-hydroxyproline was abolished by addingl-proline to the culture medium, thereby restoring normal metabolism. These results support the hypothesis that angiogenesis is regulated by collagen production and suggest that the size of newly formed microvessels is influenced by the degree of collagenization of the extracellular matrix.     

Effect of ascorbic acid on secreted proteoglycans from rabbit articular chondrocytes

Addition of ascorbic acid (25, 50 100 micrograms/ml) to cultures of rabbit articular chondrocytes did not change the total amount of proteoglycans produced. However, it induced an increased retention of these macromolecules in the pericellular fraction. The size of the proteoglycan subunits and the length of glycosaminoglycan chains, released in the medium, were not modified on exposure to ascorbic acid (25 micrograms/ml). On the other hand, the rate of non-sulfated chondroitin was increased 2.5-fold, whereas chondroitin-4-sulfate was depressed 1.5-fold.     

Regulation of collagen production and collagen mRNA amounts in fibroblasts in response to culture conditions.

Collagen synthesis and mRNA amounts for the alpha 1 and alpha 2 polypeptide chains of Type I collagen were measured in embryonic-chick tendons and in tendon cells both in suspension and in primary cultures. The percentage of protein production represented by collagen in suspension-cultured cells was initially the same as in the intact tendon; however, on an hourly basis, there was actually a steady decline in collagen production by suspended cells. Collagen production in primary cultures of chick tendon fibroblasts was decreased when compared with intact tendon, even though ascorbate-supplemented primary cultures were able to maintain higher rates of collagen production than were non-supplemented cultures. The amounts of mRNA for alpha 1(I) and alpha 2(I) polypeptide chains of collagen responded in similar fashions to different culture conditions and were compared with the amounts of mRNA for beta-actin. In primary cultures the available alpha 1 and alpha 2 collagen mRNAs support proportionately higher collagen production than in the intact tendon. However, the ratio of alpha 1/alpha 2 mRNA and polypeptide-chain synthesis did not remain 2:1, but increased with the concomitant production of Type I trimers composed of three alpha 1 chains. Removal of fibroblasts from their environment in vivo appears to alter the amounts of mRNA for alpha 1 and alpha 2 chains and to alter the utilization of those mRNAs for polypeptide synthesis.     

The role of TGF-β in the pathogenesis of primary open-angle glaucoma

Transforming growth factor-β2 (TGF-β2) is found in increasing amounts in aqueous humor and reactive optic nerve astrocytes of patients with primary open-angle glaucoma (POAG), a major cause of blindness worldwide. The available data strongly indicate that TGF-β2 is a key player contributing to the structural changes in the extracellular matrix (ECM) of the trabecular meshwork and optic nerve head as characteristically seen in POAG. The changes involve an induction in the expression of various ECM molecules and are remarkably similar in trabecular meshwork cells and optic nerve head astrocytes. The ECM changes in the trabecular meshwork most probably play a role in the increase of aqueous humor outflow resistance causing higher intraocular pressure (IOP). In the optic nerve head, TGF-β2-induced changes might contribute to deformation of the optic nerve axons causing impairment of axonal transport and neurotrophic supply and leading to their continuous degeneration. The increase in IOP further adds mechanical stress and strain to optic nerve axons and accelerates degenerative changes. In addition, high IOP might induce the expression of activated TGF-β1 in trabecular meshwork cells and optic nerve head astrocytes; this again might significantly lead to the progress of axonal degeneration. The action of TGF-β2 in POAG is largely mediated through the connective tissue growth factor, whereas the activities of TGF-β1 and -β2 are modulated by the blocking effects of bone morphogenetic protein-4 (BMP-4) and BMP-7, by gremlin that inhibits BMP signaling and by several species of microRNAs.     

Changes in the synthesis of minor cartilage collagens after growth of chick chondrocytes in 5-bromo-2'-deoxyuridine or to senescence

Analyses were made of the minor collagens synthesized by cultures of chondrocytes derived from 14-day chick embryo sterna. Comparisons were made between control cultures, cultures grown for 9 days in 5-bromo-2'-deoxyuridine (BrdU) and clones of chondrocytes grown to senescence. Separation of minor collagens from interstitial collagens was achieved by differential salt precipitation in the presence of carrier collagens in acid conditions. The precipitate at 0.9 M NaCl 0.5 M acetic acid from control cultures was shown by CNBr peptide analysis to contain only the alpha 1(II) chain of type II collagen, whereas after BrdU treatment or growth to senescence synthesis of only alpha 1(I) and alpha 2(I) chains occurred. The synthesis of type III collagen was not detected. Analysis of the precipitate at 2.0 M NaCl, 0.5 M HAc from control cultures demonstrated the synthesis of 1 alpha, 2 alpha and 3 alpha chains together with the synthesis of short chain (SC) collagen of Mr 43000 after pepsin digestion. After BrdU treatment or growth to senescence alpha chains were isolated which possessed the migration positions on polyacrylamide gel electrophoresis (PAGE), or the elution positions on CM-cellulose chromatography, of the alpha 1(V) and alpha 2(V) chains of type V collagen. In addition, for BrdU-treated but not for control cultures, intracellular immunofluorescent staining was observed with a monoclonal antibody which specifically recognizes an epitope present in the triple helix of type V collagen. Synthesis of short chain (SC) collagen was not detected after BrdU treatment or growth to senescence. These results suggest that chick chondrocytes grown in conditions known to cause switching of collagen synthesis from type II to type I collagen also undergo a switch from the synthesis of 1 alpha, 2 alpha and 3 alpha chains to the synthesis of the alpha 1(V) and alpha 2(V) chains of type V collagen. It appears that there are several cartilage-specific collagens which together undergo a regulatory control to the synthesis of collagens typical of other connective tissues.     

Overexpression of myocilin in cultured human trabecular meshwork cells

The trabecular meshwork, a specialized eye tissue, is a major site for regulation of the aqueous humor outflow. Malfunctioning of the trabecular meshwork is believed to be responsible for development of glaucoma, a blinding disease. Myocilin is a gene linked to the most common form of glaucoma. Its expression is known to be upregulated by glucocorticoids in trabecular meshwork cells and the altered myocilin level may be the culprit for glaucomatous conditions such as corticosteroid-induced glaucoma. In this study, we examined the influence of myocilin overexpression on the adhesion, spreading, migration, phagocytosis, and apoptosis of human trabecular meshwork cells in culture. When the myocilin expression was increased by 3- to 4-fold, the transfectants showed a dramatic loss of actin stress fibers and focal adhesions. Cell adhesion to fibronectin and spreading were also compromised. Myocilin thus appeared to have a de-adhesive activity, similar to that reported extensively with matricellular proteins. The transfected cells in addition displayed an increased sensitivity to apoptosis. These results demonstrate that overexpression of myocilin renders trabecular meshwork cells in a de-adhesive and vulnerable state. This vulnerability may be the basis for pathologic consequences in subtypes of glaucoma.     

Mechanisms of ATP release, the enabling step in purinergic dynamics

The only effective intervention to slow onset and progression of glaucomatous blindness is to lower intraocular pressure (IOP). Among other modulators, adenosine receptors (ARs) exert complex regulation of IOP. Agonists of A(3)ARs in the ciliary epithelium activate Cl(-) channels, favoring increased formation of aqueous humor and elevated IOP. In contrast, stimulating A(1)ARs in the trabecular outflow pathway enhances release of matrix metalloproteinases (MMPs) from trabecular meshwork (TM) cells, reducing resistance to outflow of aqueous humor to lower IOP. These opposing actions are thought to be initiated by cellular release of ATP and its ectoenzymatic conversion to adenosine. This view is now supported by our identification of six ectoATPases in trabecular meshwork (TM) cells and by our observation that external ATP enhances TM-cell secretion of MMPs through ectoenzymatic formation of adenosine. ATP release is enhanced by cell swelling and stretch. Also, enhanced ATP release and downstream MMP secretion is one mediator of the action of actin depolymerization to reduce outflow resistance. Inflow and outflow cells share pannexin-1 and connexin hemichannel pathways for ATP release. However, vesicular release and P2X(7) release pathways were functionally limited to inflow and outflow cells, respectively, suggesting that blocking exocytosis might selectively inhibit inflow, lowering IOP.