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Masters Thesis Final_Chang.pdf (33.39 MB)

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Using MicroRNAs 146a and 155 to Mitigate Barotrauma and Atelectrauma in Simulated Ventilator-Induced Lung Injury

Chang, Christopher J
http://rave.ohiolink.edu/etdc/view?acc_num=osu1524188199388787

Abstract Details

2018, Master of Science, Ohio State University, Biomedical Engineering.
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are lung disorders characterized by increased permeability of the alveolar barrier, resulting in fluid buildup and hypoxia. Patients with ALI or ARDS often require mechanical ventilation to displace occluded fluid and restore blood oxygenation. However, mechanical ventilation exposes alveolar and small airway epithelial cells to abnormal mechanical forces, which can exacerbate lung inflammation and injury, known as ventilator-induced lung injury (VILI). MicroRNAs, short RNAs with post-translational regulatory roles in gene expression, have emerged as promising therapeutic targets to protect against VILI. MicroRNAs 146a and 155 have been implicated in innate immunity, and shown to modulate inflammatory response during simulated lung injury . Delivery of microRNA cargos is critical for clinical translation into future VILI therapy. Endogenous extracellular vesicles (EVs) have emerged as potential drug carriers capable of delivering microRNAs of interest. To study EV-mediated delivery of microRNA-146a, A549 epithelial cell or differentiated THP-1 macrophage monocultures were incubated with either EVs containing pre-miR-146a or scramble gene, or reduced-serum media for 24 hours. MicroRNA expression levels were evaluated via qRT-PCR. EVs delivered pre-miR-146a into A549 and THP-1 cell cocultures, then oscillatory pressure (20 cmH2O, 0.2Hz) was applied for 16 hours. Secretion of interleukin (IL)-1ß, IL-6, and IL-8 was quantified via ELISA. MicroRNA-146a was overexpressed in monocultures of A549 and PMA-differentiated THP-1 cells. In cocultures with applied oscillatory pressure, dampening of IL-1ß and IL-6 secretion was inconclusive. Secretion of IL-8 significantly increased between pressure and no-pressure groups, with EVs potentially increasing pro-inflammatory response. Relative fold-change in cytokine secretion between treatment groups did not change. Overexpression of microRNA-146a has previously been shown to regulate pro-inflammatory response to oscillatory pressure. This work shows that EVs have then potential to deliver pre-miR-146a into A549 epithelial cells and differentiated THP-1 alveolar macrophages. Further work characterizing and optimizing EV drug carriers is needed to demonstrate its translational value in modulating VILI in ALI/ARDS patients. To simulate cyclic fluid-filled airway reopening in VILI, A549 cells, transfected with pre-miR-155 or scramble gene, were grown to confluence on 40mm glass cover slips coated with type I collagen and loaded into a Bioptechs parallel plate flow chamber. Five semi-infinite bubbles were passed over the cells. Cell injury/death was quantified via Live/Dead staining. This experiment was repeated with lipopolysaccharide (LPS) treatment to model endotoxic conditions. Cellular response to microRNA-155 overexpression was evaluated using qRT-PCR, Western blot and f-actin staining techniques. A549 epithelial cells overexpressing microRNA-155 exhibited significantly reduced lung injury/death compared to epithelial cells transfected with a scramble gene. This result was not seen under LPS-treatment conditions. Western blot probes for RhoA, myosin light-chain kinase, and control GAPDH, and f-actin staining were inconclusive in determining a mechanism by which microRNA-155 alters cell mechanics or morphology. MicroRNA-155 may be a promising therapeutic target in ALI/ARDS patients, as microRNA-155 overexpression significantly reduced epithelial cell injury/death in simulated VILI. This result did not hold up in a two-hit model of VILI under endotoxic conditions. Uncovering the mechanistic effect of overexpressing microRNA-155 in A549 cells requires further research.
Samir Ghadiali (Advisor)
Joshua Englert (Committee Member)
112 p.
Biomedical Engineering
lung injury; acute lung injury; acute respiratory distress syndrome; ventilator-induced lung injury; microRNA; miR-155; miR-146a; atelectrauma; barotrauma; extracellular vesicle; lung epithelial cell; alveolar macrophage;

Recommended Citations

Citations

  • Chang, C. J. (2018). Using MicroRNAs 146a and 155 to Mitigate Barotrauma and Atelectrauma in Simulated Ventilator-Induced Lung Injury [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1524188199388787

    APA Style (7th edition)

  • Chang, Christopher. Using MicroRNAs 146a and 155 to Mitigate Barotrauma and Atelectrauma in Simulated Ventilator-Induced Lung Injury. 2018. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1524188199388787.

    MLA Style (8th edition)

  • Chang, Christopher. "Using MicroRNAs 146a and 155 to Mitigate Barotrauma and Atelectrauma in Simulated Ventilator-Induced Lung Injury." Master's thesis, Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1524188199388787

    Chicago Manual of Style (17th edition)

osu1524188199388787
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This open access ETD is published by The Ohio State University and OhioLINK.