Defense Date

2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Pharmacotherapy and Outcomes Science

First Advisor

Donald F. Brophy

Second Advisor

Gretchen M. Brophy

Third Advisor

Ramesh Natarajan

Fourth Advisor

Bruce Spiess

Fifth Advisor

Jurgen Venitz

Abstract

Introduction: Neutrophils (PMNs) and Macrophages are the first responders recruited consecutively to the site of injury/inflammation. PMNs’ response/fate as well as macrophage reprogramming ultimately determine the course of resolution of inflammation. Physiologic wound healing has a significant inflammatory component. An exaggerated inflammation however is self-defeating leading to delayed healing. Parenteral vitamin C (VitC) attenuated inflammation in murine sepsis models and in patients with sepsis. However information about the mechanisms by which VitC regulates these events is limited.

Methods: Humanized mice lacking VitC synthesis capability (Gulo-/-) were used. VitC sufficient and deficient mice were challenged with sterile inflammation, or septic insults. Some VitC deficient mice received parenteral VitC (200mg/kg) following the challenge to give deficient + AscA mice up to 14 days. Using a murine model of excisional wound, two full thickness excisional wounds were created on the back of the different Gulo-/- mice groups. Wound tissues were excised at day 7 and 14 post-wounding for analysis. Cell counts, immunohistochemistry, circulating free DNA, the expression of pro- and anti-inflammatory proteins were investigated. Additional in vitro experiments were carried out using human PMN (huPMNs), THP-1 monocyte/macrophage, and neonatal human dermal fibroblasts (HnDF).

Results: VitC deficiency delayed resolution of lung inflammation and led to exaggerated pro-inflammatory responses. PMNs from VitC deficient mice demonstrated increased autophagy, histone citrullination, and NFκB activation, while inhibiting apoptosis. VitC sufficiency/supplementation restored macrophage phenotype, as well as attenuated neutrophil extracellular trap (NET) formation. VitC attenuated pro-inflammatory responses in THP-1 macrophages. In wound healing model, wounds from VitC sufficient/AscA infused mice had lower gene expression of the pro-inflammatory mediators; higher expression of genes promoting wound healing and resolution. Exposure of HnDF to AscA increased their intracellular VitC levels; promoted fibroblast proliferation and induced expression of fibroblast self-renewal genes.

Conclusion: Our findings identify VitC as a novel regulator of PMN and macrophage responses. In wound healing, VitC favorably impacted the spatiotemporal expression of transcripts associated with early resolution of inflammation and tissue remodeling. Collectively, these results substantiate the protective notion of parenteral VitC and support its clinical use.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

4-27-2015

Available for download on Wednesday, April 22, 2020

Share

COinS