Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.

This integrated approach clearly demonstrates that DEX treatment increases TM cell stiffness concurrent with elevated αSMA expression and activation of the mitogen-activated protein kinase (MAPK) pathway, stiffens the ECM in vitro along with upregulation of Wnt antagonists and fibrotic markers embedded in a more organized matrix, and increases the stiffness of TM tissues in vivo. These results demonstrate glucocorticoid treatment can initiate the biophysical alteration associated with increased resistance to aqueous humor outflow and the resultant increase in IOP.

The rabbit is commonly used to evaluate new corneal prosthetics and study corneal wound healing. Knowledge of the stiffness of the rabbit cornea would better inform design and fabrication of keratoprosthetics and substrates with relevant mechanical properties for in vitro investigations of corneal cellular behavior. This study determined the elastic modulus of the rabbit corneal epithelium, anterior basement membrane (ABM), anterior and posterior stroma, Descemet’s membrane (DM) and endothelium using atomic force microscopy (AFM). In addition, three-dimensional collagen fiber organization of the rabbit cornea was determined using nonlinear optical high-resolution macroscopy. Elastic modulus as determined by AFM for each corneal layer was: epithelium 0.57 ± 0.29 kPa (mean ± SD), ABM 4.5 ± 1.2 kPa, anterior stroma 1.1 ± 0.6 kPa, posterior stroma 0.38 ± 0.22 kPa, DM 11.7 ± 7.4 kPa, and endothelium 4.1 ± 1.7 kPa. Biophysical properties, including elastic modulus, are unique for each layer of the rabbit cornea and are dramatically softer in comparison to the corresponding regions of the human cornea. Collagen fiber organization is also dramatically different between the two species with markedly less intertwining observed in the rabbit versus human cornea. Given that substratum stiffness considerably alters corneal cell behavior, keratoprosthetics that incorporate mechanical properties simulating the native human cornea may not elicit optimal cellular performance in rabbit corneas that have dramatically different elastic moduli. These data will allow for the design of substrates that better mimic the biomechanical properties of the corneal cellular environment.

PURPOSE. Primary open-angle glaucoma is characterized by increased resistance to aqueous humor outflow and a stiffer human trabecular meshwork (HTM). Two Yorkie homologues, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif, encoded by WWTR1 (TAZ), are mechanotransducers of the extracellular-microenvironment and coactivators of transcription. Here, we explore how substratum stiffness modulates the YAP/TAZ pathway and extracellular matrix genes in HTM cells and how this may be play a role in the onset and progression of glaucoma.METHODS. HTM cells from normal donors were cultured on hydrogels mimicking the stiffness of normal (5 kPa) and glaucomatous (75 kPa) HTM. Changes in expression of YAP/ TAZ related genes and steroid responsiveness were determined. Additionally, transglutaminase-2 expression was determined after YAP silencing.RESULTS. YAP and TAZ are both expressed in human trabecular meshwork cells. In vitro, YAP and TAZ were inversely regulated by substratum stiffness. YAP and 14-3-3r were downregulated to different extents on stiffer substrates; TAZ, tissue transglutaminase (TGM2), and soluble frizzled-related protein-1 (sFRP-1) were significantly upregulated. CTGF expression appeared to be altered differentially by both YAP and TAZ. Myocilin and angiopoietin-like 7 expression in response to dexamethasone was more pronounced on stiffer substrates. We demonstrated a direct effect by YAP on TGM2 when YAP was silenced by small interfering RNA.CONCLUSIONS. The expression of YAP/TAZ and ECM-relatedgenes is impacted on physiologically relevant substrates. YAP was upregulated in cells on softer substrates. Stiffer substrates resulted in upregulation of canonical Wnt modulators, TAZ and sFRP-1, and thus may influence the progression of glaucoma. These results demonstrate the importance of YAP/TAZ in the HTM and suggest their role in glaucoma. (Invest Ophthalmol Vis Sci.

Redox state sustained by reactive oxygen species (ROS) is crucial for regeneration; however, the interplay between oxygen (O2), ROS and hypoxia-inducible factors (HIF) remains elusive. Here we observe, using an optic-based probe (optrode), an elevated and steady O2 influx immediately upon amputation. The spatiotemporal O2 influx profile correlates with the regeneration of Xenopus laevis tadpole tails. Inhibition of ROS production but not ROS scavenging decreases O2 influx. Inhibition of HIF-1α impairs regeneration and stabilization of HIF-1α induces regeneration in the refractory period. In the regeneration bud, hypoxia correlates with O2 influx, ROS production, and HIF-1α stabilization that modulate regeneration. Further analyses reveal that heat shock protein 90 is a putative downstream target of HIF-1α while electric current reversal is a de facto downstream target of HIF-1α. Collectively, the results show a mechanism for regeneration via the orchestration of O2 influx, ROS production, and HIF-1α stabilization.