Synthetic polymeric substrates as potent pro‐oxidant versus anti‐oxidant regulators of cytoskeletal remodeling and cell apoptosis

Abstract

The role of reactive oxygen species (ROS)-mediated cell signal transduction pathways emanating from engineered cell substrates remains unclear. To elucidate the role, polymers derived from the amino acid L-tyrosine were used as synthetic matrix substrates. Variations in their chemical properties were created by co-polymerizing hydrophobic L-tyrosine derivatives with uncharged hydrophilic poly(ethylene glycol) (PEG, Mw = 1,000 Da), and negatively charged desaminotyrosyl-tyrosine (DT). These substrates were characterized for their intrinsic ability to generate ROS, as well as their ability to elicit Saos-2 cell responses in terms of intracellular ROS production, actin remodeling, and apoptosis. PEG-containing substrates induced both exogenous and intracellular ROS production, whereas the charged substrates reduced production of both types, indicating a coupling of exogenous ROS generation and intracellular ROS production. Furthermore, PEG-mediated ROS induction caused nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase and an increase in caspase-3 activity, confirming a link with apoptosis. PEG-rich pro-oxidant substrates caused cytoskeletal actin remodeling through beta-actin cleavage by caspase-3 into fractins. The fractins co-localized to the mitochondria and reduced the mitochondrial membrane potential. The remnant cytosolic beta-actin was polymerized and condensed, events consistent with apoptotic cell shrinkage. The cytoskeletal remodeling was integral to the further augmentation of intracellular ROS production. Conversely, the anti-oxidant DT-containing charged substrates suppressed the entire cascade of apoptotic progression. We demonstrate that ROS activity serves an important role in "outside-in" signaling for cells grown on substrates: the ROS activity couples exogenous stress, driven by substrate composition, to changes in intracellular signaling. This signaling causes cell apoptosis, which is mediated by actin remodeling.

High-content image informatics of the structural nuclear protein NuMA parses trajectories for stem/progenitor cell lineages and oncogenic transformation

“…While elucidating the underlying mechanisms is beyond the scope of this study, we hypothesize that the biomaterials modulate exogenous and intracellular reactive oxygen species (ROS) levels, and thus influence oncogenesis [41–43]. In an earlier study, we showed that PEGcontaining substrates induce ROS production, whereas DT-containing polymers have the opposite effect [19]. Alternatively, electrostatic binding of nickel ions to the DT polymer surface may reduce nickel bioavailability and toxicity.…”

“…Tyrosine-derived polycarbonates [17], polymethacrylates [18], and poly(L-lactic acid) (Resomer L-206; Boehringer Ingelheim; Ridgefield, CT) were spin-coated onto cover glass using established protocols [19]. MSCs were seeded on polymer-coated substrates at a density of 20,000 cells per cm 2 , prior to NiSO 4 treatment, for either 3 or 11 days.…”

Modular polymer design to regulate phenotype and oxidative response of human coronary artery cells for potential stent coating applications

“…Cells were purchased from Cell Applications, Inc. (San Diego, CA). Cells were cultured for three days on test polymer samples before end point experiments in order to evaluate short-term response to polymer test substrates following the previous studies [3, 4]. …”

“…Balance of cellular oxidative mechanisms is crucial for maintaining vascular homeostasis and preventing pathogenesis; aberrant ROS activities have been implicated in several cardiovascular diseases including hypertension and atherosclerosis [11–15]. Polymer composition has been shown to influence ROS activity [3, 4] and we therefore chose to focus on intracellular ROS levels to evaluate how oxidative activity is involved in the maintenance of healthy SMC and EC behavior.…”

“…Three subunits were copolymerized at varying molar ratios: poly(ε-caprolactone) (PCL) is a slow degrading, hydrophobic, highly biocompatible polymer that has been used in various biomedical applications [2, 16, 17]; poly(ethylene glycol) (PEG) is a hydrophilic polyether that can influence surface chemistry related to anti-adhesion of proteins and cells [18], bulk and degradation properties [19, 20] and oxidative activity [3, 4]; and, carboxyl PCL (cPCL) which carries a negative charge that improves hydrophilicity, can counteract repellent effects of PEG [4], and provides a site for functionalization of bioactive molecules to the polymer backbone [21]. A representative library of copolymers was synthesized and characterized, and thin film samples were prepared as culture substrates for primary HCASMCs and HCAECs as a short-term evaluation of the applicability of these polymers for future use as a coronary stent coating material.…”