Cystatin A, a Potential Common Link for Mutant Myocilin Causative Glaucoma

Myocilin (MYOC) is a 504 aa secreted glycoprotein induced by stress factors in the trabecular meshwork tissue of the eye, where it was discovered. Mutations in MYOC are linked to glaucoma. The glaucoma phenotype of each of the different MYOC mutation varies, but all of them cause elevated intraocular pressure (IOP). In cells, forty percent of wild-type MYOC is cleaved by calpain II, a cysteine protease. This proteolytic process is inhibited by MYOC mutants. In this study, we investigated the molecular mechanisms by which MYOC mutants cause glaucoma. We constructed adenoviral vectors with variants Q368X, R342K, D380N, K423E, and overexpressed them in human trabecular meshwork cells. We analyzed expression profiles with Affymetrix U133Plus2 GeneChips using wild-type and null viruses as controls. Analysis of trabecular meshwork relevant mechanisms showed that the unfolded protein response (UPR) was the most affected. Search for individual candidate genes revealed that genes that have been historically connected to trabecular meshwork physiology and pathology were altered by the MYOC mutants. Some of those had known MYOC associations (MMP1, PDIA4, CALR, SFPR1) while others did not (EDN1, MGP, IGF1, TAC1). Some, were top-changed in only one mutant (LOXL1, CYP1B1, FBN1), others followed a mutant group pattern. Some of the genes were new (RAB39B, STC1, CXCL12, CSTA). In particular, one selected gene, the cysteine protease inhibitor cystatin A (CSTA), was commonly induced by all mutants and not by the wild-type. Subsequent functional analysis of the selected gene showed that CSTA was able to reduce wild-type MYOC cleavage in primary trabecular meshwork cells while an inactive mutated CSTA was not. These findings provide a new molecular understanding of the mechanisms of MYOC-causative glaucoma and reveal CSTA, a serum biomarker for cancer, as a potential biomarker and drug for the treatment of MYOC-induced glaucoma

Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings

Consensus recommendation for mouse models of ocular hypertension to study aqueous humor outflow and its mechanisms

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings

Three-Decade Evaluation of Cerebrospinal Fluid Pressure in Open-Angle Glaucoma at a Tertiary Care Center

Elevated intraocular pressure (IOP) is the most prevalent risk factor for primary open-angle glaucoma. However, IOP alone does not fully describe a mechanical basis for disease in patients with normal tension glaucoma or primary open-angle glaucoma. The translaminar pressure difference (TLPD) theory proposes that the pressure gradient generated by the difference of IOP and cerebrospinal fluid pressure (CSFp) acting at the level of the optic nerve can lead to cupping and glaucoma when IOP is higher than normal and/or CSFp is lower than normal. The study results to date have generally supported the TLPD theory; however, varying methods, populations, and sample sizes make it difficult to compare results. To further assess whether there is an association between low CSFp and open-angle glaucoma, 30 years of clinical data that assess 96,543 lumbar punctures were analyzed. Patients with open-angle glaucoma showed a significantly lower CSFp than randomly selected normal control patients (9.9 ± 3 mm·Hg (n = 86) versus 12.1 ± 3.6 mm·Hg (n = 114), p<0.001) following adjustment for age and sex. This retrospective study provides strong evidence for an association between open-angle glaucoma and low CSFp

Perfusion of His-Tagged Eukaryotic Myocilin Increases Outflow Resistance in Human Anterior Segments in the Presence of Aqueous Humor

PURPOSE. A previous study by the authors has shown that recombinant myocilin purified from a prokaryotic expression system increases outflow resistance in cultured human anterior segments. The present study was performed to determine whether full-length myocilin purified from a human trabecular meshwork cell expression system alters outflow resistance after infusion into human anterior segments. METHODS. A feline immunodeficiency virus vector encoding both full-length myocilin (amino acids 1-503 fused to C-terminal V5 and six-histidine epitopes) and puromycin resistance was used to transduce a transformed trabecular meshwork cell line (TM5). Stably expressing cells were selected with puromycin. Recombinant myocilin was purified from the media using nickel ion affinity chromatography. Control purifications were performed on media from parental TM5 cells. Anterior segments of human eyes were placed in organ culture and perfused with either Dulbecco&apos;s modified Eagle&apos;s medium (DMEM) or DMEM supplemented with 50% porcine aqueous humor. One eye received an anterior chamber exchange with recombinant myocilin (2 g/mL), whereas the fellow eye received an equal volume of control. Immunohistochemistry was performed with anti-myocilin and anti-V5 antibodies. Native polyacrylamide gel electrophoresis was used to analyze myocilin complex formation in porcine aqueous humor. RESULTS. Recombinant myocilin in porcine aqueous humor increased outflow resistance in cultured human anterior segments (91% Ϯ 68% [mean Ϯ SD] versus 18% Ϯ 31% in fellow control eye; n ϭ 9, P ϭ 0.004). Maximum outflow resistance was obtained 5 to 17 hours after infusion and remained above baseline for Ͼ3 days. Recombinant myocilin also increased outflow resistance in eyes incubated in DMEM, but only if myocilin was preincubated with porcine aqueous humor (78% Ϯ 77% when preincubated in DMEM containing porcine aqueous humor versus 13% Ϯ 15% when preincubated with DMEM alone, n ϭ 6, P ϭ 0.03). Recombinant myocilin appears to form a complex in porcine aqueous humor with a heat-labile protein(s). Immunohistochemistry revealed the presence of myocilin in the juxtacanalicular region of the trabecular meshwork. CONCLUSIONS. Myocilin purified from human trabecular meshwork cells increased outflow resistance in cultured human anterior segments, but only after incubation with porcine aqueous humor. Recombinant myocilin appears to form a complex in porcine aqueous humor that enables it to bind specifically within the trabecular meshwork. (Invest Ophthalmol Vis Sci