Date of Award

1-1-2023

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biological Sciences

Content Description

1 online resource (xii, 219 pages) : illustrations (some color)

Dissertation/Thesis Chair

Melinda Larsen

Committee Members

J. Andrew Berglund, Paolo Forni, Morgan Sammons, Bijan Dey

Keywords

Ductal ligation, Endothelial cell, Fibrosis, Gli1, Regeneration, Salivary gland, Salivary glands

Subject Categories

Biology

Abstract

Injury and disease can contribute to excess accumulation of extracellular matrix, which if persistent, results in fibrosis, impeding proper organ function. The salivary gland can develop fibrosis following irradiation treatment for cancer therapy, Sjögren’s Disease, and other causes; however, it is unclear which stromal cells and signals participate in injury responses and disease progression and which cells may be important for restoring proper organ function. Two stromal populations that are implicated in injury and repair are the endothelial cells, lining the inner surface of blood vessels, and pericytes, that wrap around the vessels. One stromal pericyte population, expressing the transcription factor Gli1, has been implicated in fibrosis of other organs through a myofibroblast trans-differentiation event, and we examined the contribution of Gli1 to fibrotic injury. To experimentally induce a fibrotic response in female murine submandibular salivary glands, we performed ductal ligation surgery. We detected a progressive fibrotic response where both extracellular matrix accumulation and actively remodeled collagen trended upwards at 7 days and significantly increased at 14 days post-ligation. We also detected an increase in macrophages, Gli1+, and PDGFRα+ cells with injury. There was little change in extracellular matrix area, remodeled collagen area, PDGFRα, PDGFRβ, endothelial cells, neurons, or macrophages in Gli1 null salivary glands following injury, suggesting that Gli1 signaling and Gli1+ cells have only a minor contribution to mechanical injury-induced fibrotic changes in the salivary gland. We used scRNA-seq to examine cell populations that expand with ligation and/or showed increased expression of matrisome genes. Pdgfra+/Pdgfrb+ stromal cell subpopulations both expanded in response to ligation, showed increased expression, and a greater diversity of matrisome genes expressed, consistent with these cells being fibrogenic. Endothelial cells have been implicated in fibrotic responses in many organs and have also been identified as an important cell population for regeneration. We examined the transcriptome of salivary gland endothelial cells during homeostasis, fibrotic injury, and regeneration to identify putative pro-fibrotic and pro-regenerative pathways. During homeostasis, salivary gland endothelial cells have a salivary gland gene signature and have interactions with many cell types, including the pericytes, which are important for maintaining vessel stability. With ligation, a small subset of endothelial cells may be directly contributing to fibrosis by undergoing an endoMT transition. Using an unbiased approach to predict changes in cell signaling following ligation, we found that endothelial cells may be acting as predicted sources of PTPRM, APRIL, and MPZ signaling and predicted targets of TNF signaling to promote fibrosis in multiple endothelial cell subpopulations. When comparing cell signaling changes from ligation to deligation, we found that endothelial cells may be acting as sources of both CXCL and EPHA signaling and also predicted targets of CXCL signaling to promote regeneration We identified increased expression of Edn1, Pdgfa, Jag1, and Vegfc following deligation which had receptors expressed by the pericytes (Edn1, Pdgfa, and Jag1 receptors), fibroblasts (Pdgfa receptor), and endothelial cells (Vegfc receptors). Defining the signaling pathways and cell differentiation events driving fibrotic responses or promoting recovery from fibrotic injury could reveal future stromal cell-based therapeutics for restoring salivary gland function.

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