Examining the effect of Reelin on vascular endothelial cell polarity
Authors: Elnathan Heidi , Diggs Breha , Baka Daniel , McCoy Sarai , NGele Charleslyne , Khakina Beryl , Hatcher Cathy 
Published: May 2023

Introduction: Vascular 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 coronary vessel formation. We showed that epicardial-specific 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. Previously, we showed 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. 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 the 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 CLASP2 in control and RELN KD cells. Results: We observed an equivalent number of leader cells in control and RELN KD HDMECs. However, 3-fold fewer RELN KD cells had the Golgi positioned in front of the nucleus versus control HDMECs. Thus, Reelin knockdown reduced the number of polarized Golgi in vascular endothelial cells. Conclusion: Our data indicates that Reelin may regulate cell migration and polarity through Golgi polarization. These data provide a plausible explanation for impairment of coronary vessel formation in Tbx5-deficient embryonic mouse hearts with reduced Reln cardiac expression. AHA 17AIREA33660773, PCOM CCDA This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.