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There are significant changes in morphology, cytoskeletal integrity, and gene regulation in Müller cells as a function of the stiffness of the substrate. Changes in local tissue elastic modulus may have a role in vitreoretinal disorders. These findings also may have implications for strategies for improved integration of retinal prosthetics, and for stem cell therapies, particularly targeting the transcriptional regulators YAP and TAZ.

In fibrosis and cancer, degradation of basement membrane (BM) and cell invasion are considered as key outcomes of a cellular transformation called epithelial-mesenchymal transition (EMT). Here, we pose a converse question - can preexisting physical defects in the BM matrix cause EMT in normal epithelial cells? On a BM-mimicking matrix of collagen-IV-coated polyacrylamide (PA) gel, we have discovered a reverse phenomenon in which preexisting defects trigger EMT in normal epithelial cells. Through spatiotemporal measurements and simulations in silico, we demonstrate that the EMT precedes cellular mechanoactivation on defective matrices, but they occur concurrently on stiff matrices. The defect-dependent EMT caused cell invasion though a stroma-mimicking collagen-I layer, which could be disabled through MMP9 inhibition. Our findings reveal that the known BM degradation caused by cellular EMT and invasion is not a one-way process. Instead, normal epithelial cells can exploit physical defects in the BM matrix to undergo disease-like cellular transformations.

To better understand if a complex process such as phagocytosis is influenced by substrate stiffness, we investigated the influence of substrate elastic modulus on phagocytosis in the retinal pigment epithelial (RPE) cell line ARPE-19. RPE cells lie on Bruch’s membrane, directly under the retina, and phagocytose the shed photoreceptor outer segments. Bruch’s membrane is known to increase in stiffness by an order of magnitude with age and thus, this study has potential relevance in explaining retinal changes in age-related macular degeneration. ARPE-19 cells were plated on laminin-coated polyacrylamide substrates of varying elastic modulus. After 14 days in culture, a solution of latex fluorescent beads suspended in PBS was placed in each well. After an incubation time of 4 hours, flow cytometry was performed to determine the number of cells that phagocytosed a bead. The number of ARPE-19 cells that phagocytosed a bead decreased continuously as a function of increasing substrate elastic modulus (p=0.0135), and this was found to be a linear relationship (slope=−0.03305 ± 0.01104, R2 =0.4726 per 10,000 cells). Our results suggest that RPE cells display decreased phagocytosis when grown on firmer substrates, and thus, RPE cells in older eyes, in which Bruch’s membrane is stiffer, may demonstrate decreased phagocytosis. Impaired phagocytosis by RPE cells may contribute to impaired metabolism of photoreceptor outer segments and to development of macular degeneration. Material stiffness may be a critical parameter in the development of neural therapies, including retinal prosthetics and stem cell therapies.

Our objective is to improve on our previous work developing thiol-containing water-soluble copolyacrylamides that form hydrogels in situ for use as vitreous substitutes. In this study, we evaluate the incorporation of acrylic acid by varying the feed ratio of acrylic acid monomer from 0 to 40 mol% in combination with acrylamide, and bis-acryloylcystamine as the reversible cross-linker. After polymerization, the formed copolymer hydrogels were reduced with dithiothreitol to cleave the disulfide cross-linkers. Purified, lyophilized copolymers were made in a concentration range of 12.5–17.5 mg/mL (polymer in deionized water) and were gelled by oxidation. Chemical, physical, optical, and rheological characterizations along with in vitro biocompatibility studies were performed using thiazolyl blue and Electric Cell–substrate Impedance Sensing. Increasing the percentage of acrylic acid caused the polymer to gel at 12.5 mg/mL as opposed to 20 mg/mL without acrylic acid. Storage modulus values covered the range of natural vitreous (1–108 Pa). Biocompatibility testing in tissue culture with retinal pigment epithelial cells (ARPE-19) showed no toxicity at 10 mg/mL or less when compared to controls, higher concentrations. In contrast to our previously reported copolyacrylamide hydrogels, these hydrogels remain optically clear and gel at lower concentrations and have the potential for use as vitreous substitutes.

Following ocular trauma and retinal detachment, gliotic changes in the retina may develop over the subsequent month, a process known as PVR (proliferative vitreoretinopathy). There have been no successful therapeutic interventions to inhibit PVR. The protein CTGF (Connective Tissue Growth Factor) has been associated with retinal PVR and other fibrotic diseases of the retina in clinical studies but the mechanistic link between different pathologies and retinal gliosis has not been determined. In addition, CTGF has been previously noted to be associated, in some cases, with YAP/TAZ (Yes-associated protein and Tafazzin protein complex), transcriptional regulatory proteins that change subcellular localization in response to mechanical cues, such as the stiffness of the underlying material. We have previously shown that the mRNA for CTGF is markedly (100-fold) upregulated in retinal Müller cells grown on soft substrates. In order to evaluate if the mechanism by which mechanotransduction modulating CTGF production in retinal Müller cells involves the YAP/TAZ complex, this study tests the influence of substrate stiffness on the time dependence of CTGF protein expression, as well as subcellular localization of YAP/TAZ using a conditionally-immortalized mouse retinal Müller cell line plated on laminin-coated, polyacrylamide substrates of varying elastic modulus. Changes were assayed using immunohistochemistry and ELISA (Enzyme-Linked ImmunoSorbent Assay). In retinal Müller cells, the relationship between elastic modulus and the pattern of CTGF protein expression was bimodal, with CTGF levels rising more rapidly for cells on hard substrates and more slowly for cells grown on soft substrates. In addition, nuclear localization of YAP/TAZ corresponded directly to the maximum CTGF expression.

ε-Aminocaproic acid (EACA) is a lysine analogue antifibrinolytic drug used to treat bleeding disorders in humans and domestic animals. Its use in zoological medicine is rare, and dosage is anecdotal. One possible application of EACA is to treat bleeding associated with prepatent Otostrongylus arteritis in Northern elephant seals ( Mirounga angustirostris ) presenting to wildlife rehabilitation centers. This study used an in vitro model of hyperfibrinolysis and a thromboelastograph-based assay to estimate the therapeutic plasma concentration of EACA in elephant seals (85 μg/ml, 95% confidence interval = 73.8-96.8 μg/ml). A concurrent pharmacokinetic study of orally administered, single-dose EACA found that doses of 75 and 100 mg/kg achieved therapeutic plasma concentrations (>85 μg/ml), but the drug was rapidly eliminated and remained in the therapeutic range for only 0.4 and 1.5 hr, respectively. Models of repeated oral dosing at 100 mg/kg every 6 hr predict that therapeutic plasma concentration will be maintained for 31.7% (7.6 hr) of a 24-hr period. More frequent dosing would be required to maintain continuous therapeutic concentrations but would be impractical in a wildlife rehabilitation setting. Further pharmacodynamic studies to evaluate the duration of action of EACA in elephant seals and a prospective, placebo-controlled study are needed to determine if EACA is effective in decreasing bleeding associated with prepatent Otostrongylus arteritis and other bleeding disorders in this species.