ORCID

https://orcid.org/0009-0000-3189-8851

Date of Award

Spring 2026

Language

English

Embargo Period

5-10-2026

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biological Sciences

Program

Biology

First Advisor

Melinda Larsen

Committee Members

Morgan Sammons, Michelle Lennartz, Scott Tenenbaum, Gabriele Fuchs

Keywords

Fibroblasts, Inflammation, Fibrosis, Proteomics, Transcriptomics

Subject Categories

Cell Biology | Developmental Biology

Abstract

Fibrosis is characterized by an accumulation of proteins in the extracellular matrix (ECM) that results in altered tissue architecture and reduced organ function. In the salivary glands, fibrosis leads to loss of saliva, dysbiosis of the oral microbiome, an increase in oral infections, difficulty in digestion of food and an overall reduction in the quality of life. Understanding the progression of fibrosis and how it affects human health is a crucial step for designing effective therapeutics. Understanding fibroblast function is integral to advancing the field due to the many roles they play in maintaining the ECM. To perform preliminary analysis, ex-vivo experiments are used to narrow down ideal therapeutic candidates. For fibrosis research, organoids serve as ideal models for initial testing due to the speed at which testing can occur and because they exhibit similar morphology and cellular diversity observed in native tissues. Organoids also allow us to introduce macrophages which are key regulators of injury responses, fluorescently labeled fibroblasts, and cells that have been subject to RNA interference. To study the development of fibrosis in-vivo, we used the ductal ligation model to induce a fibrotic phenotype in mouse submandibular salivary glands. We paired this surgery with single cell RNA sequencing (scRNA-seq) to study transcriptomic changes within the stromal compartment in the tissue. ScRNA-seq analysis revealed that fibroblasts responded to the injury with an initial inflammatory cascade 1 day after injury which led to increased expression of extracellular matrix regulators, secreted factors and collagens 3 days post injury. After 7 days, there was an increase in expression of proteoglycan and glycoprotein expression. Transcriptomic data allows us to predict the proteome of salivary glands, but it has its limitations. To amend this, we performed mass spectrometry on decellularized salivary glands to enrich for ECM bound proteins. Using male and female mice, we see the similarities and sex-based differences of salivary glands and injury responses in mice. An analysis of the matrix related proteins revealed that male glands inherently contain more ECM proteins at homeostasis but become like female salivary glands after ductal ligation. The role of the Transforming Growth Factor Beta (TGFβ) signaling pathway is known to be a master regulator of injury response and tissue remodeling. Using transgenic mice, we can study the role this pathway plays in fibroblast function by selectively knocking out (KO) the key receptor TGFβR2 in fibroblasts. We compared the fibrotic response in salivary glands of control wild type (WT) mice with that of mice in which we removed exon 4 of the Tgfbr2 gene prior to surgery specifically in fibroblasts using the transgenic model Pdgfra creERT2;Tgfbr2fl/fl . We evaluated the tissue at various timepoints post-ligation using immunohistochemistry (IHC), Picrosirius Red staining, and mass spectroscopy. We were able to see the decrease in fibrillar protein expression in TGFβR2 KO mice, and our proteomic analysis provided insight into proteins that are absent in KO mice. Using various methods, we see that ligated salivary glands have an increase in expression of myofibroblast markers and Tgfbr2 in fibroblasts. Removal of this receptor leads to significant changes in injury response and tissue architecture after ductal ligation. We also see that although male and female mice exhibit different proteomes at homeostasis, they respond to injury in a similar fashion. We determined that ductal ligation of mouse salivary glands induces a TGFβ-dependent fibrotic response which follows a pattern of protein expression at different timepoints. We identified ECM proteins that are increase with fibrosis and manipulation of genes downstream of TGFβ signaling is a potential therapeutic avenue.

License

Creative Commons Attribution 4.0 International License
This work is licensed under a Creative Commons Attribution 4.0 International License.

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