NYMC Student Theses and Dissertations

Date of Award

8-30-2021

Document Type

Doctoral Dissertation - Restricted (NYMC/Touro only) Access

Degree Name

Doctor of Philosophy

Department

Biochemistry and Molecular Biology

First Advisor

Austin M. Guo

Second Advisor

Sachin A. Gupte

Third Advisor

Thomas M. Jeitner

Abstract

Compensatory angiogenesis is an important adaptation for the recovery from critical ischemia. The newly formed blood vessels, however, often fail to adequately perfuse damaged tissues leading to chronic and life-threatening disease. Consequently, much effort has and continues to be expended to understand and regulate this response. We recently identified 20-hydroxyeicosatetraenoic acid (20-HETE) as a key contributor of ischemia-induced angiogenesis. However, the precise cellular and molecular mechanisms by which ischemia causes these 20-HETE increases remain completely unknown. Here we address the hypothesis that inflammatory neutrophil-derived myeloperoxidase (MPO) and hypochlorous acid (HOCl) promote 20-HETE production which critically contributes to ischemia-induced angiogenesis.

In our attempts to investigate the role of immunity in this pathway, we assessed post-ischemic 20-HETE production and angiogenesis due to femoral artery ligation in immunocompetent Balb/C mice as compared to severe combined immunodeficiency (SCID) mice. We found that 20-HETE production, measured by LC/MS/MS, failed to increase in the affected hindlimb gracilis muscles of SCID mice compared to gracilis muscles of the immunocompetent mice. This deficiency in 20-HETE production is accompanied with a decreased angiogenic phenotype, determined by laser doppler perfusion imaging (LDPI) and microvascular density counts (MVD). With further investigation, we demonstrated that these changes are due to the actions of neutrophils. Neutrophil-depletion by lymphocyte antigen 6 complex locus G6D/6CD (Ly6g/c) also leads to a deficiency in 20-HETE production and angiogenesis in these ischemic tissues. These decreases are not evident in the ischemic gracilis muscles of mice depleted of macrophages using clodronate liposomes. MPO is a major neutrophil protein responsible for many of the actions of neutrophils. To examine the possibility that neutrophils contribute to 20-HETE production and angiogenesis through MPO, we assayed post-ischemic angiogenic changes and 20-HETE production in global MPO-knockout (MPO-/-) mice. MPO-/- mice exhibit a remarkable absence of post-ischemic 20-HETE production accompanied by decreased angiogenesis. Furthermore, we showed that both MPO and HOCl are markedly increased in hindlimb gracilis muscles post-ischemia using immunofluorescence microscopy and in vivo HOCl live imaging.

Next, we examined the role of MPO and HOCl in the induction of 20-HETE production in human dermal microvascular endothelial cells (HDMEC): the primary effector cells in angiogenesis. We found that catalytically active MPO and HOCl both induce the expression of the human 20-HETE synthase cytochrome P450 A11 (CYP4A11) at the transcriptional and translational level in HDMEC. The increases in CYP4A11 expression are complemented with increased 20-HETE production. In our studies, we also determined that HOCl significantly stimulates hypoxia inducible factor-1α (HIF-1α) transcriptional activity and protein expression. Importantly, pharmacological interference of HIF results in the ablation of HOCl-induced CYP4A11 transcripts, as well as a significant reduction in CYP4A11 protein.

Taken together, these observations support a model whereby neutrophils are recruited to hypoxic sites of injury, induce HIF-1α and CYP4A11, and thereby ensure the production of 20-HETE and nascent blood vessels. The implications of this study suggest promising targets for the treatment of 20-HETE related disorders and diseases related to neovascularization. Depletion of neutrophils to attenuate these neovascular processes results in neutropenia, which in turn results in serious and potentially dangerous infections. Also, neutrophil recruitment and activation are fundamental components of inflammation as well as normal vascular reactivity and function. However, MPO deficiency is common and rarely life-threatening (1 in 3000 people in North America). Thus, the use of safe and effective MPO inhibitors (Verdiperstat), represent remarkable therapeutic value in the treatment of vascular disorders and cancers.

Available for download on Monday, January 31, 2022

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