Dissertation
Soft saves: the protective effects of a soft substrate against cytoxicity through reactive oxygen species reliant mechanisms
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2020
DOI: 10.17077/etd.005375
Abstract
The extracellular matrix (ECM), a complex network of proteins and polysaccharides, serves to shelter cells and facilitate intercellular communication. Cell behavior can be regulated through the ECM by both mechanical and biochemical interactions. These biochemical signals are a common focus of research, but our understanding of the role of mechanical signals lags behind. Multiple studies have shown a strong relationship between ECM stiffness and cell differentiation, motility, actin polymerization, and morphology. However, there is very little information on the effects of ECM stiffness on toxicity.
With polyacrylamide gels, the environmental stiffness the cell experienced was regulated to that of physiologically relevant levels. This allowed the study of both the role and mechanism of ECM stiffness in cell behavior.
In order to understand the relationship between ECM stiffness and cytotoxicity, silica nanoparticles were chosen as a model toxin due to a well-established mechanism of toxicity through reactive oxygen species (ROS). SH-SY5Y cells were chosen for their sensitivity to substrate stiffness and for their well understood response to silica nanoparticle exposure. Undifferentiated SH-SY5Y human neuroblastoma cells were exposed to amorphous 50 nm silica nanoparticles wherein it was observed that cells on a soft substrate exhibited a higher retention of cell viability compared to those on a stiff substrate or on glass. It was similarly observed that cells on a softer substrate had lower ROS generation and retained their mitochondrial membrane potential despite cells on the softer substrate endocytosing more silica than their compatriots on the stiff substrate and the same amount as those on glass. It was also observed that SH-SY5Y cells exposed to silica nanoparticles exhibited a dose-dependent cell stiffening as well as changes in cell morphology and actin cytoskeletal structure.
A similar pattern of survival on soft substrates was seen when SH-SY5Y cells, human primary cortical neurons (HPCNs), or BV2 cells were exposed to chlorpyrifos on a soft substrate compared to a stiff substrate. Chlorpyrifos was used due to its ability to cause ROS mediated cell death and due to its current risk as an environmental health risk. BV2 cells and HPCNs were used due to the highly deleterious effects of chlorpyrifos in the brain in vivo. Exposure to chlorpyrifos also induced changes in cell morphology, actin cytoskeletal structure, and cell stiffening in SH-SY5Y and HPCNs as measured by atomic force microscopy. BV2 cells demonstrated cell softening with exposure to chlorpyrifos as well as a more rounded cell morphology and limited actin cytoskeletal changes. ROS levels increased in SH-SY5Y cells exposed to chlorpyrifos, but no observable changes in ROS levels were seen in BV2 cells. However, treatment with NAC protected against chlorpyrifos toxicity in BV2 cells, suggesting that ROS were involved in the observed cytotoxicity. Cytochalasin D and blebbistatin offered mild protection against chlorpyrifos induced toxicity in SH-SY5Y, while Y-27632 did not ameliorate the cytotoxicity. In BV2 cells, Y-27632 protected completely and cytochalasin D offered mild protection against chlorpyrifos, but blebbistatin offered no protection against cytotoxicity.
This research indicates that the ECM stiffness plays a crucial, biophysical role in guiding cell survival in regards to the cytotoxicity of silica nanoparticles and chlorpyrifos in these cell lines, and demonstrates that the mechanisms through which this occurs is highly likely to be cell specific protection against ROS. This project will help focus future research into better in vitro models for cytotoxicity that incorporate substrate stiffness and research into the mechanisms of stiffness-dependent cell behavior which occur with aging and with many diseases.
Details
- Title: Subtitle
- Soft saves: the protective effects of a soft substrate against cytoxicity through reactive oxygen species reliant mechanisms
- Creators
- Kendra Joan Bell
- Contributors
- Lewis Stevens (Advisor)Ethan Anderson (Committee Member)Jonathan Doorn (Committee Member)Aliasger Salem (Committee Member)Alexei Tivanski (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Pharmacy
- Date degree season
- Spring 2020
- DOI
- 10.17077/etd.005375
- Publisher
- University of Iowa
- Number of pages
- xv, 147 pages
- Copyright
- Copyright 2020 Kendra Joan Bell
- Language
- English
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references (pages 123-140).
- Public Abstract (ETD)
The extracellular matrix (ECM) is a network of proteins and other molecules that serves to shelter and guide cells. The ECM controls for behavior through both physical and chemical properties. While there has been a great deal of research on the influence of chemical changes on cells, our understanding of the role of physical and mechanical changes is not as well defined. When the ECM changes its mechanical properties, getting stiffer or softer, it can drastically affect the motility, shape, and uptake of cells. However, there has been minimal research on how changes in the mechanical properties of the ECM affects toxicity in cells.
The use of gels mimicking the stiffness of tissues in the body allowed study of the role of ECM stiffness in toxicity. In multiple cell types and with two different tools used to induce cell death through reactive oxygen species, we determined that toxicity was dependent upon stiffness. Cells on a softer substrate consistently had higher survival. Meanwhile, we observed that both a stiffer ECM and the introduction of a toxic substance induced cell stiffening and changes in cell shape. Inhibitors for different cell mechanisms allowed us to determine that the way the ECM affects cell behavior depends on both the cell type and the toxin. In conclusion, it was found that softening the cell’s environment protected the cell against toxicity.
- Academic Unit
- Pharmacy; Craniofacial Anomalies Research Center
- Record Identifier
- 9983949695802771
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