Examining the Effects of Reelin on Vascular Endothelial Cell Polarity

Date of Award

7-2023

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Cathy Hatcher

Second Advisor

Minal Mulye

Third Advisor

Ruth Borghaei

Abstract

Introduction: Vascular endothelial cells differentiate into motile leader cells and stalk cells, become polarized, and migrate toward growth factor signals during vessel formation. Our goal is to shed light on mechanisms contributing to the effect of Reelin on coronary vessel formation. Previous data from the Hatcher lab showed that epicardia-lspecific deletion of the Tbx5 transcription factor in embryonic mouse hearts impaired coronary vessel formation and reduced cardiac expression of the RELN gene encoding Reelin extracellular matrix glycoprotein. Reelin is expressed in coronary vascular endothelial cells of embryonic mouse hearts. We silenced endogenous Reelin expression in human dermal microvascular endothelial cells (HDMECs) with small interfering RNAs (siRNA) designed to target human RELN and evaluated its role in these cells. Additionally, we found that RELN knockdown (KD) in HDMECs reduced cell migration. Cell polarity is a key to vascular endothelial cell migration, and polarization of the Golgi is vital for cell polarity. Reelin is known to modulate the Golgi in neuronal cells by affecting phosphorylation of the cytoplasmic linker associated protein CLASP2, a downstream effector of GSK3β. Because CLASP2 is an upstream effector of microtubule reorganization in neuronal and endothelial cells, we hypothesize that RELN KD leads to CLASP2 phosphorylation and prevents polarization of vascular endothelial cells by reducing the association between Golgi-derived microtubules and actin filaments to inhibit cell migration.

Study Objective: The purpose of this study was to examine the role of Reelin signaling in regulating vascular endothelial cell polarity to initiate migration during angiogenesis.

Methods: We assayed the impact of Reelin on vascular endothelial cell polarity by conducting scratch wound assays on control or RELN KD HDMECs. Cells were allowed to migrate into a denuded region created by the scratch. After a 6-hour migration period, HDMECs were fixed and stained for immunofluorescence with GM130 cis-Golgi marker and counterstained with DAPI to visualize the nucleus. Leader cells localized to the leading edge in the migration front of the scratch wound. We determined leader cell polarity by assessing the position of the Golgi relative to the nucleus. Also, we assessed GSK3β and CLASP2 expression in control and RELN KD cells.

Results: Although we confirmed that knockdown of Reelin delays vascular endothelial cell migration, we did not observe a significant difference in Golgi positioning between RELN KD and control cells. Results of western blot analysis did not show significant difference between the ratio of active GSK3β and inactive phospho-GSK3β in RELN KD and control cells.

DISCUSSION: Our data indicates that Reelin may not be a direct regulator of cell migration and polarity through Golgi polarization. However, Reelin may influence cell migration indirectly through its ability as a signal transduction molecule to inactivate CLASP2 which is known to regulate cell polarity and migration. We may gain insight into how Reelin regulates migration of vascular endothelial cells through direct analysis of CLASP2 phosphorylation. In the future, studies should be designed to focus on the molecular effects of Reelin on regulation of vascular endothelial cell migration to elucidate its role in coronary vessel formation.

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