Graduation Year

2022

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

George E. Davis, M.D., Ph.D.

Committee Member

Jerome Breslin, Ph.D.

Committee Member

Joshua Scallan, Ph.D.

Committee Member

Mack Wu, M.D.

Keywords

3D, Basement Membrane, Endothelial Cell, Pericyte, Regression, Serum-Free

Abstract

Capillary formation and maturation can only occur through a series of highly orchestrated events that requires the coordinated efforts of both endothelial cells (ECs) and pericytes. While ECs are perfectly capable of creating tube networks on their own, the maturation and stabilization of these tube networks require the presence of a second cell type, pericytes, to create stable capillary networks. Previous studies have identified factors required for pericyte recruitment to EC tube networks, however, we hypothesized that there were still unaccounted for EC-derived molecules that were contributing to pericyte recruitment. In the first part of this study, we used serum-free, 3D (3-dimensional) assays to identify PDGF (platelet-derived growth factor)-B, PDGF-DD, ET (endothelin)-1, TGF (transforming growth factor)-β, and HB-EGF (heparin-binding EGF-like growth factor) as EC-derived molecules contributing to pericyte recruitment. When these molecules are blocked in combination using either neutralizing antibodies or pharmacologic inhibitors, we see pericyte recruitment reduced to less than 10% of that in controls. In addition, pericyte blockade causes a loss of most basement membrane components, as well as EC tube networks to become wider and less branched.

While capillary formation has been one of our interests for many years, recent studies have identified IL (interleukin)-1β, TNF (tumor necrosis factor) α, and thrombin as key regulators of capillary regression, causing selective death of the endothelium, while retaining pericytes. Observing that the pericytes were quickly changing morphology, becoming larger in 3D cultures, and knowing others have previously linked pericytes to fibrosis, we hypothesized these pericytes were becoming myofibroblasts. Using serum-free, 3D collagen and fibrin matrices, along with a novel stable capillary wall model, we demonstrate IL-β, TNFα, and thrombin leading to capillary regression. After 1 week, we show pericyte-induced collagen I deposition in the ECM (extracellular matrix), which is noticeably absent from control conditions. Additionally, we observe loss of the basement membrane.

Finally, we speculated that something must be working to prevent blood capillary regression in our bodies. Our hypothesis was that lymphatic capillaries would be responsible for clearing proinflammatory mediators from the interstitial fluid. As such, we wanted to see if there were any proinflammatory mediators that would cause lymphatic capillary regression. Using novel, serum-free, 3D collagen and fibrin assays, we identify TNFα, IFN (interferon) γ, and thrombin as potent inducers of lymphatic tube network regression. We then identify pharmacologic agents to rescue LEC tube network regression.

on this work, we speculate that TNFα, IFNγ, and thrombin cause lymphatic capillary regression, followed by blood capillary regression due to accumulation of IL-β, TNFα, and thrombin, followed by pericyte induced collagen I deposition, eventually leading to fibrosis.

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