Date of Award

Spring 4-5-2023

Embargo Period

4-20-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Pathobiology

College

College of Graduate Studies

First Advisor

Amy Bradshaw

Abstract

Antecedent conditions that affect the heart, such as aortic stenosis (AS), can develop into left ventricular pressure overload (LVPO). LVPO is associated with increased myocardial interstitial fibrosis, specifically fibrillar collagen, that leads to increased myocardial stiffness. Patients undergoing surgical aortic valve replacement (SAVR) to correct AS demonstrate significant but incomplete regression of fibrosis. Although current therapies normalize hemodynamic load, treatments that regress collagen content within the myocardium remain a critical need. To elucidate cellular mechanisms of this persistent fibrosis in LVPO, we utilized a transverse aortic constriction (TAC) and removal of the constriction (unTAC) murine model. Outputs were assessed for: Control, 2wk TAC, 4wk TAC, 4wk TAC+2wk unTAC, 4wk TAC+4wk unTAC, and 4wk TAC+6wk unTAC. Collagen volume fraction (CVF), collagen hybridizing peptide (CHP), and macrophage abundance were assessed by immunohistochemistry. Macrophage phenotype was assessed by flow cytometry and rt-qPCR. Cytokine profiles and enzymes implicated in collagen turnover were determined by immunoassay and immunoblots. To determine the role of macrophages in collagen turnover following alleviation of LVPO, macrophages were depleted with clodronate liposomes at time of unTAC surgery and endpoints were measured at 2wk unTAC. CHP reactivity was significantly increased at 2wk unTAC compared to other time points. A significant reduction in CVF was observed at 4wk unTAC compared to 4wk TAC and 2wk unTAC although it remained increased compared to control levels. A defined temporal pattern in myocardial macrophage cell count was observed: increased in 2wk TAC that decreased at 4wk TAC followed by a further increase at 2wk unTAC and then decreased at 4wk and 6wk unTAC. Macrophage counts at all time points remained higher than control. Macrophage area was significantly increased at 2wk unTAC compared to all other time points. Profibrotic macrophage markers, F4/80+CD206+, F4/80+CD163+, and Ly6ClowF4/80high, were increased in TAC compared to unTAC. Depletion of macrophages reduced CHP reactivity and decreased Cathepsin K and proMMP2 levels. In conclusion, normalization of hemodynamic load leads to regression of cardiomyocyte hypertrophy but does not result in complete regression of myocardial fibrosis. Temporal changes in macrophage cell count and phenotype play a critical role in the development of fibrosis during TAC and the partial, but incomplete regression of fibrosis in unTAC.

Rights

Copyright is held by the author. All rights reserved.

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