Jose M. Gonzalez

Functional properties of fibronectin in the trabecular meshwork

Fibronectin plays a number of important roles in the extracellular matrix (ECM) including providing structural support and signaling cues for cell survival, migration, differentiation, gene expression, growth factor signaling, and cell contractility. In this review, we examine recent findings about the biological and structural properties of fibronectin and discuss how these properties could contribute to the regulation of aqueous humor (AH) outflow in the trabecular meshwork (TM).

Identification of the Active Site in the Heparin II Domain of Fibronectin that Increases Outflow Facility in Cultured Monkey Anterior Segments

PURPOSE To determine the active site in the Heparin II (HepII) domain of fibronectin that regulates outflow facility in cultured anterior segments and disrupts the actin cytoskeleton in transformed human trabecular meshwork (TM-1) cells. METHODS Outflow facility was determined by two-level, constant-pressure perfusion in cultured anterior segments of rhesus and cynomolgus monkey eyes. One segment from each pair was exchanged with either the HepII domain or an integrin/syndecan binding peptide (IDAPS or PPRARI) from the HepII domain. To assay changes in the actin cytoskeleton, TM-1 cells were incubated for 24 hours with or without the HepII domain, PPRARI, or IDAPS. Changes were monitored with phase and immunofluorescence microscopy. RESULTS HepII domain (100 microg/mL) and PPRARI (500 microg/mL) increased outflow facility by 31% +/- 13% (n = 9, P < 0.05) and 24% +/- 9% (n = 8, P < 0.05), respectively in cultured anterior segments after an overnight infusion. Perfusion with IDAPS (500 microg/mL) had no effect on outflow facility. In TM-1 cultures, 250 microg/mL of the HepII domain or 4 mg/mL of PPRARI disrupted the assembly of actin filaments. A lower concentration of PPRARI (2 mg/mL) disrupted the actin cytoskeleton when used in combination with a nondisrupting concentration of the HepII domain (30-60 microg/mL). In contrast, IDAPS did not disrupt the actin cytoskeleton under any condition tested. CONCLUSIONS The active site in the HepII domain that regulates outflow facility in cultured anterior segments and disrupts the actin cytoskeleton in TM-1 cells is the syndecan/integrin binding sequence, PPRARI.

Analyzing Live Cellularity in the Human Trabecular Meshwork

PURPOSE To directly visualize the live cellularity of the intact human trabecular meshwork (TM) and quantitatively analyze tissue viability in situ. METHODS Human donor corneoscleral rims were sectioned immediately before intravital dye incubation to label nuclei (Hoechst 33342 & propidium iodide [PI]); cytosol (CellTracker Red CMTPX, calcein AM); and membranes (octadecyl rhodamine B chloride [R18]), followed by 2-photon microscopy. Viability was assessed by counting cells in tissue colabeled with PI and Calcein AM. Some tissues were exposed to Triton X-100 to establish dead tissue controls. Fresh postmortem eyes (within 48 hours of death) represented viable tissue controls. Tissues with live cellularity exceeding 50% were considered viable. RESULTS Hoechst nuclear labeling was seen throughout the TM, among the autofluorescent beams, plate-like structures and fibers of the meshwork, and within tissue gaps and pores. CellTracker-labeled live cells were attached to autofluorescent TM structures and filled corneoscleral meshwork pores. R18-labeling revealed the membrane distributions of interconnected cells. Calcein-positive cells were visible in all TM layers, but not in tissues killed by Triton X-100 exposure. Dead control tissues showed PI staining in the absence of Calcein-positive cells. Two-thirds of the standard donor tissues we received possessed viable TM, having a mean live cellularity of 71% (n = 14), comparable with freshly postmortem eyes (76%; n = 2). Mean live cellularity of nonviable tissue was 11% (n = 7). CONCLUSIONS We have visualized and quantified the live cellularity of the TM in situ. This provided unique perspectives of live cell-matrix organization and a means of assaying tissue viability.

In Situ Autofluorescence Visualization of Human Trabecular Meshwork Structure

PURPOSE To characterize the three-dimensional (3-D) structure of the human trabecular meshwork (TM) by two-photon excited (TPEF) autofluorescence (AF) and optical sectioning without conventional histologic embedding and sectioning. METHODS Viable human ex vivo explants of the anterior chamber angle containing the aqueous humor drainage tissue in situ were imaged by TPEF to localize AF and Hoechst 33342 nuclear fluorescence. An autofluorescent marker in Schlemms Canal (SC) aided SC situ visualization. En face and orthogonal views of the TM were generated. RESULTS In the innermost uveal TM, AF signals outlined an intricate 3-D network of fine branching beams with large openings between the beams. In the adjacent corneoscleral TM, beams were thicker and coalesced as plate-like structures with pore-like openings. Linear and coiled AF fibers were visible on the background AF of beams. Deeper, in the external TM, this organization changed to fine fiber arrays orientated in the tissues longitudinal axis, reminiscent of the cribriform plexus of the juxtacanalicular TM (JCT). In the outermost JCT, AF of fine fibers was sparse, then undetectable as optical sections approached the inner wall of SC. Cell nuclei were closely associated with the TM structural extracellular matrix. CONCLUSIONS We have used TPEF and optical sectioning to exploit AF as a useful method to visualize the structure of the human conventional aqueous drainage pathway in situ. Ancillary nuclear staining allowed cell association with the AF structures to be seen. This approach revealed a unique 3-D perspective of the TM that is consistent with known TM structural characteristics.

Tissue-Based Imaging Model of Human Trabecular Meshwork

We have developed a tissue-based model of the human trabecular meshwork (TM) using viable postmortem corneoscleral donor tissue. Two-photon microscopy is used to optically section and image deep in the tissue to analyze cells and extracellular matrix (ECM) within the original three-dimensional (3D) environment of the TM. Multimodal techniques, including autofluorescence (AF), second harmonic generation (SHG), intravital dye fluorescence, and epifluorescence, are combined to provide unique views of the tissue at the cellular and subcellular level. SHG and AF imaging are non-invasive tissue imaging techniques with potential for clinical application, which can be modeled in the system. We describe the following in the tissue-based model: analysis of live cellularity to determine tissue viability; characteristics of live cells based on intravital labeling; features and composition of the TMs structural ECM; localization of specific ECM proteins to regions such as basement membrane; in situ induction and expression of tissue markers characteristic of cultured TM cells relevant to glaucoma; analysis of TM actin and pharmacological effects; in situ visualization of TM, inner wall endothelium, and Schlemms canal; and application of 3D reconstruction, modeling, and quantitative analysis to the TM. The human model represents a cost-effective use of valuable and scarce yet available human tissue that allows unique cell biology, pharmacology, and translational studies of the TM.

Intraocular pressure measurement in acepromazine‐sedated mice

trexate because of adverse effects, and adalimumab and certolizumab failed to control her symptoms so she was started on mycophenolate mofetil (MM). Although MM resulted in quiescence of uveitis and improved her macular oedema, it did not control the psoriatic lesions, and therefore, GLM was added to her regimen. On the current regimen of GLM and MM, patient achieved complete resolution of macular oedema, and uveitis remained quiet with exception of one flare that resolved quickly with topical steroids for 1 month. Patient later developed rapidly progressive cataract OD despite the satisfactory control of uveitis, resulting in decreased VA. GLM was discontinued after 9 months because of insufficient control of arthritis. In this study, we present outcomes of GLM treatment in three patients with SpA-associated uveitis. There are reports of GLM therapy for juvenile idiopathic arthritis and uveitis syndrome (JIA) uveitis, Behçet uveitis, human leukocyte antigen -B27 (HLA-B27)-associated uveitis and idiopathic retinal vasculitis (summary presented in Table 2). To our best knowledge, this is the first report of GLM treatment in patients with ASand PsAassociated uveitis. In contrast with the prior report about GLM treatment in HLA-B27 associated uveitis, our case series details response of both systemic and eye symptoms to GLM and its related adverse effects. While on GLM, patients 1 and 2 achieved satisfactory control of their ocular inflammation, but only patient 1 also achieved systemic disease control. For patient 3, uveitis was well controlled on GLM and MM regimen with complete resolution of macular oedema. However we cannot attribute these effects only to GLM as the patient was receiving another immunosuppressant simultaneously. This patient’s decreased vision was secondary to cataract progression that could be due to longstanding inflammation and/or previous long-term steroid use. Patient 3 did not achieve systemic disease control with GLM. Two of the three patients tolerated GLM well despite having adverse reactions to other anti-TNF agents, and one patient developed side-effects with all TNF inhibitors including GLM. This report suggests that GLM can be effective treatment for controlling uveitis in SpA patients who may or may not have responded to other TNF-inhibitors. We acknowledge that this is a small case series and further studies with longer follow up are required to better evaluate the efficacy and safety of GLM in treatment of SpA-associated uveitis.

Observing live actin in the human trabecular meshwork.

The actomyosin network of trabecular meshwork (TM) cells influences intraocular pressure (IOP) and aqueous humor drainage resistance1 and represents an important therapeutic target for glaucoma. The biology of actin in the TM and effect of agents that alter actin have been studied primarily in cultured TM cells. We are developing a tissue-based model of the human TM in which live cells and cellular interactions can be directly observed in situ.2,3 Here we report our initial novel observations of actin in live cells within the human TM following in situ baculovirus transduction with actin-RFP. Human donor corneoscleral tissue was received in Optisol GS (Bausch & Lomb, Rochester, NY). For institutional reasons, age, death and other patient information were not available to us. Typical age at death ranged from 40-70 years and typical post-mortem age was 7-days (oral communication, Dr. Martin Heur), with experiments begun within a day of receipt.2-5 TM was cut into segments (Fig. 1) and representative segments were randomly selected for viability analysis, as previously described,2 prior to incubations for F-actin labeling. Briefly, tissue was co-incubated with Calcein AM and propidium iodide at 37°C and 8% CO2 prior to live cell imaging. Tissues with at least 50% Calcein-positive cells were considered viable.2 Viable tissue was incubated with Cellular Lights™ Actin-RFP (Life Technologies; n=5) following manufacturers instructions. Cellular Lights uses a baculovirus delivery vector (BacMam technology) that transduces mammalian cells and directs fluorescence expression by TagRFP fusion to the N-terminus of beta-actin. Some specimens were co-incubated with Hoechst 33342 to label cell nuclei. For comparison, different tissue segments were fixed (4% parformaldehyde), permeabilized in 5% Triton X-100 (2h, 4°C), and incubated with Alexa Fluor 568®-conjugated phalloidin (n=40).4 Figure 1 A: Location of trabecular meshwork (TM) in human corneoscleral tissue. Bar=1mm. B: Examples of wedges cut from corneoscleral donor tissue. Hashed lines indicate the anterior and posterior borders of the TM. Blood is present in Schlemms canal, immediately ... The tissue was imaged on a PerkinElmer™ Ultraviewer spinning disk confocal microscopy system with 63× water immersion objective. Excitation/emission: 488/525nm (autofluorescence); 555/584nm (Actin-RFP; phalloidin) and 350/460nm (Hoechst) Following baculovirus transduction, cell clusters expressing actin-RFP (red fluorescence) were seen associated with autofluorescent TM uveal beams (Fig. 2A), corneoscleral pores (Fig. 2B,C) and juxtacanalicular fibers (Fig. 2D). Actin-RFP had a primarily cortical distribution and outlined cell borders, comparable with phalloidin labeling (compare figs. 2E-H). Actin distribution in the cytosol was perinuclear (Figs. 2D, 2H; closed arrowheads), punctate (Figs. 2A, 2C, 2D-H; open arrowheads) and filamentous (Figs. 2B-D; open arrows). In some sections, actin filaments were aligned along uveal beams (Figs. 2A, 2E) and corneoscleral pores (Figs. 2B, 2F). Some cell borders had an appearance resembling membrane ruffles typically seen in cultured cells (Fig. 2B, 2C; closed arrows). These ruffle-like structures were not observed in phalloidin-labeled cells. Nuclei were closely associated with fluorescence-labeled actin (Figs. 2A, 2D-H; asterisks). No nuclear fragmentation was seen. Figure 2 Clusters of live TM cells expressing Actin-RPF (red; A-D) or fixed, phalloidin-labeled (red) TM cells in the uveal (A, E), corneoscleral (B, C, F, G) and juxtacanalicular (D, H) regions. Membrane ruffle-like structures (closed arrows) were apparent in ... We have observed the actin cytoskeleton of live cells in the human TM following baculovirus transduction with actin-RFP. Optical sections captured various aspects of the actin cytoskeleton at different TM depths. Actin distribution was perinuclear, punctate, filamentous, and prominent in cell cortices and borders. Notably, prominent stress fibers were not seen. This may be due to the tissue micro-environment that differs from that of rigid-surfaced 2D culture; lack of serum or endogenous factors that enhance actin polymerization; or optical sectioning of cells in 3D tissue that masks stress fibers. Alternatively, the lack of uveal and posterior tissue attachments in donor tissue rims could result in decreased tensions across the TM, and explain the lack of stress fibers. Actin-RFP labeling showed similarities with phalloidin-labeled actin with one caveat. Actin-RFP revealed the presence of membrane protrusions reminiscent of ruffles that were not evident in fixed and permeabilized phalloidin-labeled cells. It could be that Actin-RFP (or GFP) labeling has particular benefits for visualizing less stable actin structures (lamellipodia, filopodia) in live cells, a possibility we plan to explore in future studies using 2-photon microscopy. We used spinning disk laser confocal microscopy that limits phototoxicity during live cell imaging. We are now optimizing our transduction protocols and using 2-photon microscopy that is less phototoxic and penetrates deeper than 1-photon microscopy. We have reported the utility of our human TM tissue-based system for live cell analysis and analysis of protein induction and expression, extracellular matrix and glaucoma markers.2-6 Our present report expands the findings of our prior studies with evidence for the models potential use in tissue-based real-time studies of actin dynamics and testing of actin-targeting glaucoma therapies.

Semi-automated vitality analysis of human trabecular meshwork

Glaucoma is associated with cell loss in the trabecular meshwork of the eye. Multiphoton microscopy aided by intravital dye labeling permits visualization of live and dead cells within the intact trabecular meshwork. We have developed a semi-automated method to quantify cellular viability within the human trabecular meshwork based on three-dimensional software-assisted tissue reconstruction and isosurface modeling. Live cellularity counts by the semi-automated method were obtained quickly and agreed with that of manual counting in the same group of tissues (1.6% group difference; n = 13), with counts in individual tissues showing a mean coefficient of variation of 10%.

Total Outflow Facility in Live C57BL/6 Mice of Different Age

Purpose: To characterize total outflow facility across the live adult mouse lifespan as a reference for mouse glaucoma studies and the common C57BL/6 background strain. Methods: Microperfusion was performed by single-needle cannulation and feedback-controlled coupling of pressure and flow to maintain a constant pressure in the anterior chambers of live C57BL/6NCrl mice aged 3-4 months (n = 17), 6-9 months (n = 10), and 23-27 months (n = 12). This mouse age range represented an equivalent human age range of young adult to elderly. We characterized the following across age groups in vivo: (1) outflow facility based on constant pressure perfusion in a pressure range of 15-35 mm Hg, (2) perfusion flow rates, and (3) anterior segment tissue histology after perfusion. Thirty-nine live mice underwent perfusion. Results: Pressure-flow rate functions were consistently linear for all age groups (all R2 > 0.96). Total outflow facility in mice aged 3-4, 6-9, and 23-27 months was 0.0066, 0.0064, and 0.0077 μL/min/mm Hg, respectively. Facility was not significantly different between age groups (all p > 0.4). The groups had closely overlapping frequency distribution profiles with right-sided tails. Post hoc estimates indicated that group facility differences of at least 50% would have been detectable, with this limit set mainly by inherent variability in the strain. A trend toward higher perfusion flow rates was seen in older mice aged 23-27 months, but this was not significantly different from that of mice aged 3-4 months or 6-9 months (p > 0.2). No histological disruption or difference in iridocorneal angle or drainage tissue structure was seen following perfusion in the different age groups. Conclusion: We did not find a significant difference in total outflow facility between different age groups across the live C57BL/6 mouse adult lifespan, agreeing with some human studies. The possibility that more subtle differences might exist ought to be judged with respect to the heterogeneity in facility at different ages. Our findings provide reference data for live perfusion studies pertaining to glaucoma involving the C57BL/6 strain.