Enzyme-induced hypoxia leads to inflammation in urothelial cells in vitro.

PURPOSE: To determine the contributions of different durations of hypoxia to NLRP3 inflammasome activation in urothelial cells and how ischemic changes in bladder tissues is an important chemical que that leads to pathological changes seen in BOO. METHODS: A rat urothelial cell line (MYP3) was exposed to either a short duration (2 h) or long duration (6 h) of enzyme-induced hypoxia. Following exposure to a short duration of hypoxia, NO and ATP concentrations were measured from supernatant media and caspase-1 levels were measured from cell lysates. In a separate experiment, cells were fixed following hypoxia exposure and immunostained for HIF-1α stabilization. RESULTS: Although short exposure of low oxygen conditions resulted in a hypoxic response in MYP3 cells, as indicated by HIF-1α stabilization and increased NO activity, NLRP3 inflammasome activation was not observed as caspase-1 activity remained unchanged. However, exposure of MYP3 cells to a longer duration of hypoxia resulted in an increase in intracellular caspase-1 activity. Furthermore, treatment with antioxidant (GSH) or TXNIP inhibitor (verapamil) attenuated the hypoxia-induced increase in caspase-1 levels indicating that hypoxia primarily drives inflammation through a ROS-mediated TXNIP/NLRP3 pathway. CONCLUSION: We conclude that hypoxia induced bladder damage requires a duration that is more likely related to elevated storage pressures/hypoxia, seen in later stages of BOO, as compared to shorter duration pressure elevation/hypoxia that is encountered in normal micturition cycles or early in the BOO pathology where storage pressures are still normal.

Stabilization of enzyme-immobilized hydrogels for extended hypoxic cell culture.

In this work, glucose oxidase (GOx)-immobilized hydrogels are developed and optimized as an easy and convenient means for creating solution hypoxia in a regular incubator. Specifically, acrylated GOx co-polymerizes with poly(ethylene glycol) diacrylate (PEGDA) to form PEGDA-GOx hydrogels. Results show that freeze-drying and reaction by-products, hydrogen peroxide, negatively affect oxygen-consuming activity of network-immobilized GOx. However, the negative effects of freeze-drying can be mitigated by addition of trehalose/raffinose in the hydrogel precursor solution, whereas the inhibition of GOx caused by hydrogen peroxide can be prevented via addition of glutathione (GSH) in the buffer/media. The ability to preserve enzyme activity following freeze-drying and during long-term incubation permits facile application of this material to induce long-term solution/media hypoxia in cell culture plasticware placed in a regular CO2 incubator.

Enzymatic Cross-Linking of Dynamic Thiol-Norbornene Click Hydrogels.

Enzyme-mediated in situ forming hydrogels are attractive for many biomedical applications because gelation afforded by the enzymatic reactions can be readily controlled not only by tuning macromer compositions, but also by adjusting enzyme kinetics. For example, horseradish peroxidase (HRP) has been used extensively for in situ crosslinking of macromers containing hydroxyl-phenol groups. The use of HRP on initiating thiol-allylether polymerization has also been reported, yet no prior study has demonstrated enzymatic initiation of thiol-norbornene gelation. In this study, we discovered that HRP can generate thiyl radicals needed for initiating thiol-norbornene hydrogelation, which has only been demonstrated previously using photopolymerization. Enzymatic thiol-norbornene gelation not only overcomes light attenuation issue commonly observed in photopolymerized hydrogels, but also preserves modularity of the crosslinking. In particular, we prepared modular hydrogels from two sets of norbornene-modified macromers, 8-arm poly(ethylene glycol)-norbornene (PEG8NB) and gelatin-norbornene (GelNB). Bis-cysteine-containing peptides or PEG-tetra-thiol (PEG4SH) were used as crosslinkers for forming enzymatically and orthogonally polymerized hydrogels. For HRP-initiated PEG-peptide hydrogel crosslinking, gelation efficiency was significantly improved via adding tyrosine residues on the peptide crosslinkers. Interestingly, these additional tyrosine residues did not form permanent dityrosine crosslinks following HRP-induced gelation. As a result, they remained available for tyrosinase-mediated secondary crosslinking, which dynamically increases hydrogel stiffness. In addition to material characterizations, we also found that both PEG- and gelatin-based hydrogels provide excellent cytocompatibility for dynamic 3D cell culture. The enzymatic thiol-norbornene gelation scheme presented here offers a new crosslinking mechanism for preparing modularly and dynamically crosslinked hydrogels.