https://www.mccormick.northwestern.edu/biomedical/markj/PDFofPapers/Johnson2006.pdf

What controls aqueous humour outflow resistance

PURPOSE. Transforming growth factor (TGF)-2 levels are elevated in glaucomatous human aqueous humor. TGF is a cytokine that alters extracellular matrix (ECM) metabolism, and excess ECM has been proposed to increase aqueous outflow resistance in the trabecular meshwork (TM) of glaucomatous eyes. This study was undertaken to investigate effects of TGF2 on secretion of fibronectin and the protease inhibitor plasminogen activator inhibitor (PAI)-1 from human TM cell cultures and perfused human ocular anterior segments. METHODS. Total RNA was isolated from pooled human TM cell monolayers and used for a gene microarray expression analysis. Supernatants from treated human TM cells were analyzed by ELISA for fibronectin or PAI-1 content. TGF2 effects on intraocular pressure (IOP) were evaluated in a perfused organ culture model using human anterior segments, and eluates were analyzed for fibronectin and PAI-1 content. RESULTS. Overnight treatment of TM cells with TGF2 upregulated multiple ECM-related genes, such as PAI-1. TGF2 also increased secretion of both fibronectin and PAI-1 from TM cells. TGF2 effects on TM cells were blocked by inhibitors of the TGF type I receptor. In perfused human anterior segments, TGF2 treatment elevated IOP and increased eluate fibronectin and PAI-1 content. CONCLUSIONS. TGF2 effects on IOP may be transduced by TGF type-I receptor-mediated changes in TM secretion of ECM-related factors such as fibronectin and PAI-1. Modulation of TGF2-induced changes in the ECM may provide a novel and viable approach to the management of glaucoma. (Invest Ophthalmol Vis Sci. 2006;47:226‐234) DOI:10.1167/iovs.05-1060

TGFβ2-Induced Changes in Human Trabecular Meshwork: Implications for Intraocular Pressure

Consensus recommendations for trabecular meshwork cell isolation, characterization and culture

The many faces of the trabecular meshwork cell.

changes in the TM that correlated with the induction of myocilin in the TM. Immunofluorescence analysis of cultured TM cells localized myocilin intracellularly in discrete perinuclear and cytoplasmic vesicular deposits as well as extracellularly on the cell surface associated with the extracellular matrix. In several DEX-treated TM cell lines, there were significant levels of myocilin secreted into the media. Enzymatic deglycosylation of proteins in the TM media converted the higher molecular weight isoforms of myocilin (;57 kDa) to the lower molecular weight isoforms (;55 kDa). CONCLUSIONS. Although the function of myocilin is unknown, induction of these TM proteins was found in eyes in which glucocorticoid-induced ocular hypertension developed. Therefore, myocilin may play an important pathogenic role in ocular hypertension in addition to its role in certain forms of POAG. (Invest Ophthalmol Vis Sci. 2001;42:1769 ‐1780)

/pdf/glucocorticoid-induction-of-the-glaucoma-gene-myoc-in-human-2fkgucs6n0.pdf

Glucocorticoid induction of the glaucoma gene MYOC in human and monkey trabecular meshwork cells and tissues.

Like corneal endothelial cells, human trabecular meshwork cells are believed to be of neural crest origin, but demonstrate physiological properties and an antithrombogenic surface similar to vascular endothelial cells. One current method for isolating trabecular meshwork cells utilizes the motile nature of these cells to migrate away from a trabecular meshwork explant in culture to more distal regions of the culture dish. This 'outgrowth' technique is limited in practice by the relatively small number of cells that migrate per explant per unit time, thus hindering the ability to gather sufficient numbers of cells for comprehensive experimentation. For this reason, we have modified an extracellular matrix digestion technique in current use for the isolation of microvascular endothelial cells to isolate human trabecular meshwork cells. This procedure is both efficient and rapid for isolating large numbers of trabecular meshwork cells and results in the availability of trabecular meshwork cells in sufficient quantities for subsequent experimentation.

Isolation and culture of human trabecular meshwork cells by extracellular matrix digestion.

Isolation and culture of human trabecular meshwork cells by extracellular matrix digestion

Purpose. Elevated intraocular pressure in those with glaucoma appears to be a function of increased resistance to movement of aqueous humor through the conventional outflow pathway. The majority of resistance in both normal and glaucomatous eyes is generated in the region between the juxtacanalicular trabecular meshwork and the inner wall of Schlemm's canal. To accommodate transient elevations in pressure, we hypothesize that conventional outflow increases rapidly due to changes in complexity of intercellular junetions between cells of the inner wall of Schlemm's canal. Methods. To test this hypothesis we examined specifically the effects of hydrostatic pressure gradients and the calcium chelator, Na 2 EDTA, on permeability of cultured human Schlemm's canal cell monolayers in isolation. Human Schlemm's Canal cells were isolated, cultured and then seeded onto permeable supports and maintained in culture to allow intercellular junctions to mature. With a minimum net transendothelial electrical resistance of 10 Ohm cm 2 , cells were placed into an Ussing-type chamber and hydraulic conductivity was calculated from pressure and flow measurements that were continuously recorded. Simultaneously, transendothelial electrical resistance was measured manually at fixed intervals. In parallel experiments, cell margins were monitored in real time by videomicroscopy. Results. During the baseline measurement period when cells were exposed to pressure but not Na 2 EDTA, hydraulic conductivity was constant but transendothelial electrical resistance decreased continuously at rate of 0.24Ω cm 2 /minute. After Na 2 EDTA treatment, no significant change in transendothelial electrical resistance was measured while, hydraulic conductivity of Schlemm's Canal monolayers increased significantly by 125%; corresponding to noticeable intercellular separations. Restoration of cell-cell contact was observed by videomicroscopy 30 minutes following washout of Na 2 EDTA and functionally after 2 hours. Conclusions. Responses of Schlemm's Canal cells to pressure and calcium chelators in vitro are consistent with a role for calcium sensitive junctions in outflow resistance in vivo.

W. D. Stamer

Papers

Isolation and culture of human trabecular meshwork cells by extracellular matrix digestion.

Isolation and culture of human trabecular meshwork cells by extracellular matrix digestion

Effect of hydrostatic pressure gradients and Na2EDTA on permeability of human Schlemm's canal cell monolayers.