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

Segmental Versican Expression in the Trabecular Meshwork and Involvement in Outflow Facility

PURPOSE. Versican is a large proteoglycan with numerous chondroitin sulfate (CS) glycosaminoglycan (GAG) side chains attached. To assess versican's potential contributions to aqueous humor outflow resistance, its segmental distribution in the trabecular meshwork (TM) and the effect on outflow facility of silencing the versican gene were evaluated. METHODS. Fluorescent quantum dots (Qdots) were perfused to label outflow pathways of anterior segments. Immunofluorescence with confocal microscopy and quantitative RT-PCR were used to determine versican protein and mRNA distribution relative to Qdot-labeled regions. Lentiviral delivery of shRNAsilencing cassettes to TM cells in perfused anterior segment cultures was used to evaluate the involvement of versican and CS GAG chains in outflow facility. RESULTS. Qdot uptake by TM cells showed considerable segmental variability in both human and porcine outflow pathways. Regional levels of Qdot labeling were inversely related to versican protein and mRNA levels; versican levels were relatively high in sparsely Qdot-labeled regions and low in densely labeled regions. Versican silencing decreased outflow facility in human and increased facility in porcine anterior segments. However, RNAi silencing of ChGn, an enzyme unique to CS GAG biosynthesis, increased outflow facility in both species. The fibrillar pattern of versican immunostaining in the TM juxtacanalicular region was disrupted after versican silencing in perfusion culture. CONCLUSIONS. Versican appears to be a central component of the outflow resistance, where it may organize GAGs and other ECM components to facilitate and control open flow channels in the TM. However, the exact molecular organization of this resistance appears to differ between human and porcine eyes. 1,2 Much of this resistance resides within the trabecular meshwork (TM) putatively within 7 to 14 m of the inner wall of Schlemm's canal in a region known as the juxtacanalicular (JCT) or cribriform region. 1-4 Since the 1950s, involvement of extracellular matrix (ECM) in outflow resistance has commonly been evoked. 1,2,5-10 There is also considerable evidence of a direct contribution to the resistance by some cell populations within this region. 1,3 More recently, synergistic interaction between the JCT and Schlemm's inner wall endothelial cells has been suggested. 2,6,12-14 Although perfusion of GAG-degrading enzymes reduces the outflow resistance in numerous species, in humans and primates, this effect has been controversial. 17-21 The identity of the specific GAGs, proteoglycans, or other ECM components that comprise outflow resistance remains unclear. 2 Both conceptually and based on several observations, versican, with supportive contributions from its attached chondroitin sulfate (CS) GAG chains and hyaluronan (HA) interactions, seems a likely contributor. 2,24 Up to 23 CS GAG side chains can be attached to these two central domains. These CS chains appear to extend away from the core protein in all directions, thus minimizing electrostatic interactions and filling large hydrodynamic volumes. Conceptually, this design is ideal to regulate movement of aqueous humor through the TM. 27 Mechanical stretching or distortion appears to be the mechanism by which TM cells sense elevated IOP and trigger IOP homeostatic responses. 30 -33 TNF␣ and IL-1␣, both of which increase outflow facility, also produce changes in versican mRNA level and isoform distribution similar t

Segmental Versican Expression in the Trabecular Meshwork and Involvement in Outflow Facility

Studies of segmental aqueous humor outflow and versican distributions suggest that this large proteoglycan has a role in outflow resistance. Effects of changing versican levels and glycosaminoglycan side chains on outflow facility support versican as an important component of outflow resistance

Inhibition of hyaluronan synthesis reduces versican and fibronectin levels in trabecular meshwork cells.

Hyaluronan (HA) is a major component of the extracellular matrix (ECM) and is synthesized by three HA synthases (HAS). Similarities between the HAS2 knockout mouse and the hdf mutant mouse, which has a mutation in the versican gene, suggest that HA and versican expression may be linked. In this study, the relationship between HA synthesis and levels of versican, fibronectin and several other ECM components in trabecular meshwork cells from the anterior segment of the eye was investigated. HA synthesis was inhibited using 4-methylumbelliferone (4MU), or reduced by RNAi silencing of each individual HAS gene. Quantitative RT-PCR and immunoblotting demonstrated a reduction in mRNA and protein levels of versican and fibronectin. Hyaluronidase treatment also reduced versican and fibronectin levels. These effects could not be reversed by addition of excess glucose or glucosamine or exogenous HA to the culture medium. CD44, tenascin C and fibrillin-1 mRNA levels were reduced by 4MU treatment, but SPARC and CSPG6 mRNA levels were unaffected. Immunostaining of trabecular meshwork tissue after exposure to 4MU showed an altered localization pattern of HA-binding protein, versican and fibronectin. Reduction of versican by RNAi silencing did not affect HA concentration as assessed by ELISA. Together, these data imply that HA concentration affects synthesis of certain ECM components. Since precise regulation of the trabecular meshwork ECM composition and organization is required to maintain the aqueous humor outflow resistance and intraocular pressure homeostasis in the eye, coordinated coupling of HA levels and several of its ECM binding partners should facilitate this process

Mapping molecular differences and extracellular matrix gene expression in segmental outflow pathways of the human ocular trabecular meshwork.

Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma, and lowering IOP remains the only effective treatment for glaucoma. The trabecular meshwork (TM) in the anterior chamber of the eye regulates IOP by generating resistance to aqueous humor outflow. Aqueous humor outflow is segmental, but molecular differences between high and low outflow regions of the TM are poorly understood. In this study, flow regions of the TM were characterized using fluorescent tracers and PCR arrays. Anterior segments from human donor eyes were perfused at physiological pressure in an ex vivo organ culture system. Fluorescently-labeled microspheres of various sizes were perfused into anterior segments to label flow regions. Actively perfused microspheres were segmentally distributed, whereas microspheres soaked passively into anterior segments uniformly labeled the TM and surrounding tissues with no apparent segmentation. Cell-tracker quantum dots (20 nm) were localized to the outer uveal and corneoscleral TM, whereas larger, modified microspheres (200 nm) localized throughout the TM layers and Schlemm's canal. Distribution of fluorescent tracers demonstrated a variable labeling pattern on both a macro- and micro-scale. Quantitative PCR arrays allowed identification of a variety of extracellular matrix genes differentially expressed in high and low flow regions of the TM. Several collagen genes (COL16A1, COL4A2, COL6A1 and 2) and MMPs (1, 2, 3) were enriched in high, whereas COL15A1, and MMP16 were enriched in low flow regions. Matrix metalloproteinase activity was similar in high and low regions using a quantitative FRET peptide assay, whereas protein levels in tissues showed modest regional differences. These gene and protein differences across regions of the TM provide further evidence for a molecular basis of segmental flow routes within the aqueous outflow pathway. New insight into the molecular mechanisms of segmental aqueous outflow may aid in the design and delivery of improved treatments for glaucoma patients

Fibronectin levels in response to 4MU treatment.

<p>(A) Fibronectin mRNA levels by qRT-PCR in TM cells treated with 0, 0.5, 1 and 2 mM 4MU for 36 hours. mRNA levels were normalized to 18S RNA levels and presented as a percentage of the control. Error bars are the s.e.m.; n = 3; * P<0.01 by one-way ANOVA. (B) Fibronectin ELISA assay to measure total fibronectin (RIPA lysates and media) protein levels in TM cells. Cells were treated for 4 days with 1 mM 4MU or vehicle-control. Error bars are the s.e.m.; n = 3. * P = 0.0001 by an unpaired Student’s t-test. (C) Western immunoblots of fibronectin protein in conditioned media from TM cells treated with vehicle-control (ctrl) or 1 mM 4MU for 3 and 4 days. The MW markers and 3 day samples are from adjacent lanes of one immunoblot and the 4 days lanes are from adjacent lanes of a different immunoblot. (D) Densitometry of fibronectin showed a significant reduction with 4MU treatment. Fibronectin levels were normalized to total protein loaded and are presented as a percentage of the control. Error bars are the s.e.m.; n = 6 for 3 days, n = 5 for 4 days. * p = 0.039 and ** p = 0.02 by one-way ANOVA. (E) Fibronectin immunostaining of TM cells treated with vehicle-control or 1 mM 4MU for 4 days and viewed by confocal microscopy. Nuclei are stained with DAPI (control) or Draq5 (4MU). Scale bars = 20 µm.</p

Versican levels in response to 4MU treatment.

<p>(A) A cell viability assay showed no significant difference (p = 0.575) in the percentage of dead cells per field between TM cells treated for 5 days with 1 mM 4MU or vehicle control. Error bars are s.e.m; n = 6 fields counted. (B) Versican mRNA levels were assessed by qRT-PCR in TM cells treated with 0, 0.5, 1 and 2 mM 4MU for 24 hours. mRNA levels were normalized to 18S RNA levels and presented as a percentage of the vehicle control. Error bars are the s.e.m.; n = 3; * p = 0.04 and ** p = 0.026 by one-way ANOVA. (C) Western immunoblots of versican in conditioned media and RIPA lysates from TM cells treated with vehicle-control (ctrl) or 1 mM 4MU for 3 and 4 days. All media lanes are from different lanes of the same immunoblot. Cell lanes are from adjacent lanes of the same immunoblot. (D) Densitometry of the V1 isoform (265 kDa) in the media showed a significant reduction with 4MU treatment at 3 and 4 days. Versican levels were normalized to total protein loaded and are presented as a percentage of the control. Error bars are the s.e.m.; n = 4 for 3 days, n = 3 for 4 days. * p = 0.031 and ** p = 0.024 by one-way ANOVA. (E) Versican immunostaining of TM cells treated with vehicle-control or 1 mM 4MU for 4 days and viewed by confocal microscopy. Inset shows a negative control with no primary antibodies. Nuclei are stained with DAPI (control and inset) or Draq5 (4MU). Scale bars = 20 µm.</p

Effect of addition of exogenous HA and hyaluronidase treatment.

<p>Confluent TM cells were treated for 2 days with 1 mM 4MU and then HA of different molecular weights (high MW = 1.5 MDa; medium MW = 700 kDa; low MW = 40 kDa) was exogenously added for a further 3 days. Versican and fibronectin protein levels in conditioned media were assessed by Western immunoblot. (A) Versican V1 isoform and (B) fibronectin protein levels are significantly decreased by 4MU treatment in all cases, but addition of 3 concentrations (100 µg/ml, 500 µg/ml and 1 mg/ml) of HA of HMW, MMW and LMW did not significantly alter levels compared to 4MU treatment alone. The Western immunoblots show data from a 500 µg/ml treatment. All lanes were from adjacent lanes of the same immunoblot. Error bars are the s.e.m.; n = 3. (C) Hyaluronidase (1.5 µg/ml; 1 unit) was added to confluent TM cells in serum-free media each day for 3 days. Western immunoblots and densitometry showed that versican V1 and fibronectin protein levels were significantly decreased by hyaluronidase treatment. All lanes were from adjacent lanes of a single immunoblot. Versican and fibronectin values were normalized to total protein loaded and are presented as a percentage of the control. Error bars are the s.e.m.; n = 4; * p = 0.03 and ** p = 0.001 by one-way ANOVA.</p

Effect of addition of glucose and glucosamine to 4MU-treated TM cells.

<p>(A) An HA ELISA assay was used to quantitate HA levels in TM cells treated with 10 mM glucosamine for 48 hrs. HA levels were significantly increased in media (*p = 0.008), RIPA lysates (**p = 0.04) and total media + RIPA lysates (***p = 0.004) compared to untreated control TM cells. Error bars are the s.e.m; n = 3. (B, C) Confluent TM cells were treated for 4 days with 1 mM 4MU alone, or 4MU with 10 mM glucose or 10 mM glucosamine in serum-free media. Versican and fibronectin protein levels in conditioned media were assessed by Western immunoblot. (B) Versican V1 isoform and (C) fibronectin protein levels were significantly decreased by 4MU treatment. Addition of 20 mM or 10 mM glucose or glucosamine did not significantly alter protein levels compared to 4MU treatment with either glucose or glucosamine supplementation. The Western immunoblots show data from a 10 mM treatment. For the versican immunoblot, all lanes were from adjacent lanes of the same immunoblot. The fibronectin immunoblot shows non-adjacent lanes from the same immunoblot. Versican and fibronectin values were normalized to total protein loaded and are presented as a percentage of the control. Error bars are the s.e.m.; n = 3.</p

The effects of 4MU on mRNA levels of other ECM genes.

<p>(A) The table summarizes the previously identified binding interactions of HA, versican and fibronectin reported in the literature. (B) Quantitative RT-PCR of mRNA levels of CD44, tenascin C, fibrillin-1, CSPG6 and SPARC in TM cells treated with 0, 0.5, 1 and 2 mM 4MU for 36 hours. All mRNA levels were normalized to 18S RNA levels and presented as a percentage of the control. Error bars are the s.e.m. N = 3, apart from CSPG6, where n = 4. * P<0.05 by one-way ANOVA.</p