Date of Award

2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cell and Molecular Pharmacology and Experimental Therapeutics

College

College of Graduate Studies

First Advisor

Peggi Angel

Second Advisor

Richard Drake

Third Advisor

Lauren Ball

Fourth Advisor

Amy Bradshaw

Fifth Advisor

Don Menick

Abstract

Congenital aortic valve stenosis (CAVS) affects up to 10% of the world population without medical therapies to treat the disease. New molecular targets are continually being sought that can halt CAVS progression, particularly in pediatric patients where bioengineered solutions are not ideal. Collagen deregulation is a hallmark of pediatric CAVS yet remains mostly undefined. Here, histological studies were paired with high resolution accurate mass (HRAM) collagen-targeting proteomics and imaging mass spectrometry to investigate collagen fiber production with localized collagen regulation associated with human AV development and pediatric end-stage CAVS (pCAVS). Histological studies identified collagen fiber realignment and unique regions of high-density collagen in pCAVS. Proteomic analysis reported specific collagen peptides are modified with hydroxylated prolines (HYP), a post-translational modification critical to stabilizing the collagen triple helix. Quantitative data analysis reported significant regulation of collagen HYP sites across patient categories, providing insight to collagen-cell receptor binding. In addition to chromatographic-based proteomic analysis, Matrix Assisted Laser Desorption Ionization imaging mass spectrometry (MALDI-IMS) methods were developed to further address the localized structure-function relationship of the extracellular matrisome in aortic valve tissue. Here, a novel serial enzyme strategy was developed to define the glycosaminoglycome, N-glycome, as well as the collagen and elastin proteome from a single tissue section for MALDI-IMS applications. These multimodal MALDI-IMS techniques could define unique matrisome profiles based off tissue hemodynamics, as well as identify collagen localization unable to be detected by tradition histopathology. Finally, as a proof-of-concept study toward biomaterials applications, MALDI-IMS was used to localize human collagen-based hydrogels within an infarcted mouse heart, as well as analyze its impact on endogenous extracellular matrix (ECM) remodeling. The studies presented in this dissertation are the first of their kind to detail the collagen types and HYP modifications associated with human AV development and pediatric CAVS. Additionally, our findings show evidence for the use of MALDI-IMS in assessing the therapeutic application of collagen-based biomaterials. We anticipate that this study will inform new therapeutic avenues that inhibit valvular degradation in pCAVS and bioengineered options for valve replacement.

Rights

All rights reserved. Copyright is held by the author.

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