Date of Award

12-2024

Document Type

Thesis

Degree Name

Master of Science in Biology

Department

Biology

First Reader/Committee Chair

Bournias-Vardiabasis, Nicole

Abstract

Squamous metaplasia (SM) of the airway epithelium is an abnormal condition that is caused by exposure to airborne toxicants, cigarette smoke, pollutants, and pathogens. Since SM of the airway was first documented in the 1970s, data on this epithelial disease remain limited. More research is needed to fully understand this epithelial disorder and to prevent its progression into severe illnesses such as chronic bronchitis (CB), COPD, and squamous cell carcinoma. We developed 3D models of SM using airway basal stem cells (ABSCs) isolated from lung obtained from postmortem human donors. The ABSCs were cultured at the air-liquid interface (ALI) for 4 weeks to differentiate and form human tracheobronchial epithelial tissues (hTET). Two methods were used to induces SM. The tissues were scratched to inflict an epithelial lesion. The epithelium responded to the scratch with epithelial hyperplasia and squamous cell metaplasia. By the 4th day after the injury, epithelial hyperplasia and SM were most severe but spontaneously resolved after 10 days, demonstrating that both conditions are reversible. The second method to induce SM in ABSCs or hTET? model involved basolateral treatments with Marlboro red (MR) cigarette smoke solution dissolved in culture medium on differentiating ABSCs for 4 weeks. Antibodies specific for ABSCs (keratin 5), cilia (acetylated tubulin), and squamous cells (involucrin) were used to identify any occurrence of SM in the tissues. For this method, ABSCs from two donors were used to produce hTETs, but only one produced tissue with SM in response to MR solution. In the donor that did not produce severe SM, the layer of ABSCs stained by keratin 5 was thicker in the cigarette smoke treated tissue than the control. Differentiation of cilia was not affected by the treatment, but involucrin concentrations were higher in the treated tissue. In contrast to donor 1, the hTETs from donor 2 exhibited clear signs of SM upon MR treatment, which included a lack of pseudostratification, absence of cilia, increase of keratin 5, and elevated involucrin concentrations. Since donor 2 ABSCs produced a better model of SM tissues, these tissues were subjected to RNA isolation and analysis via reverse transcription-qPCR, and protein isolation and analysis via Western blot. Primers for TINCR, a long non-coding RNA associated with involucrin, were used to quantify TINCR expression in donor 2 hTETs treated with MR solution, which increased TINCR in a concentration dependent manner. Additionally, proteins downstream of TINCR (involucrin, PAK4, JAG, and NOTCH) were quantified in the lysates of treated donor 2 hTETs. Involucrin concentrations were elevated, consistent with the histology data. PAK4 concentrations increased, while JAG and NOTCH decreased when donor 2 hTETs were treated with MR. These changes in protein concentrations confirmed that MR treatment increased TINCR expression, which controlled the rise in involucrin concentration. These data will be useful for further SM model development to better understand SM, and eventually prevent its progression into incurable airway diseases.

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