Congenital coronary artery anomalies occur in up to 5% of the human population. Such anomalies can lead to myocardial ischemia, myocardial infarction and sudden death. Coronary vessels are composed of endothelial and smooth muscle cells. The primary function of vascular endothelial cells is to provide a barrier between the vessel lumen and surrounding tissue. Vascular repair processes often recapitulate developmental mechanisms. Elucidation of signaling mechanisms that govern formation of nascent coronary endothelial cells is required to develop therapeutic strategies aimed at regrowth of congenitally malformed or damaged coronary vessels. Embryonic mice with epicardial‐specific deletion of Tbx5 (Tbx5epi−/−) exhibit delayed epicardial cell attachment to the myocardium and impaired coronary vessel formation. This leads to punctate cardiac hemorrhaging that may stem from deficits in vascular integrity. Expression of Reln mRNA, that encodes the Reelin extracellular matrix glycoprotein, is also reduced in embryonic Tbx5epi−/− mouse hearts. Reelin localizes to some, but not all, coronary vascular endothelial cells during mammalian cardiogenesis. These findings lead us to speculate that expression of Reelin in coronary vascular endothelial cells may be critical for establishing vascular integrity.Study ObjectiveThe goal of this study was to elucidate contributions of Reelin to endothelial cell function for establishing vascular integrity during coronary vessel formation.MethodsWe utilized human microvascular endothelial cells (HMECs) to assess contributions of Reelin to endothelial cell function as these cells are appropriate in vitro models of vessel formation and have endogenous Reelin expression. We achieved RELN gene silencing through a small interfering RNA‐mediated approach that led to >90% reduction in both RELN mRNA and Reelin protein expression in comparison to negative control‐transfected HMECs. Next, control and RELN‐silenced endothelial cells were subjected to physiological assays to assess their ability to establish vascular integrity. First, we examined the ability of RELN‐silenced HMECs to adhere to the basement membrane through exposure of these cells to various matrices. Second, we assessed cell membrane permeability of RELN‐silenced HMECs that were plated as a monolayer on a collagen‐coated semi‐permeable barrier. This served as an indicator of in vitro vascular permeability. The cell monolayer was treated with a permeability factor and fluorescent‐labeled Dextran was added on top of the monolayer. Fluorescent Dextran passed through the cell monolayer at a rate proportional to membrane permeability. Permeability was measured by quantifying the fluorescence of the media on the basolateral side of the monolayer.ResultsOur results indicate that RELN silencing alters in vitro endothelial cell adhesion and cell membrane permeability.ConclusionsWe conclude that Reelin plays a critical role during coronary vessel development as it establishes vascular integrity through maintenance of cell adhesions and membrane permeabilitySupport or Funding InformationAHA 17AIREA3360773 and PCOM Center for Chronic Disorders of AgingThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

IntroductionEstablishment of the coronary circulation is a critical step in heart development. Formation of functional coronary vessels is required for the heart to undergo successful compaction of the ventricular myocardium. Oxygen and nutrients are delivered to the contractile myocardium through the coronary vasculature. Molecular signaling pathways that control formation of the coronary vessels are being identified. Immunofluorescent studies from our lab indicate that the Reelin extracellular matrix glycoprotein is expressed within the vascular endothelial cells of developing and mature coronary vessels of the mouse heart. The Reelin signaling pathway is known to contribute to brain and lymphatic system development by influencing neuronal and endothelial cell behaviors such as adhesion and migration. However, its role in coronary vascular development is as yet unidentified.Study ObjectiveThe goal of this study was to elucidate the potential roles of Reelin in formation of the coronary vasculature by identifying how it regulates vascular endothelial cell behaviors. We hypothesized that Reelin signaling regulates establishment of coronary vessel integrity during cardiogenesis by influencing vascular endothelial cell adhesion as well as cell membrane permeability and resistance.MethodsWe utilized the primary human dermal microvascular endothelial cell line (HDMEC) as an in vitro system to examine contributions of Reelin to vascular endothelial cell behavior. A small interfering RNA‐mediated approach was used to silence the endogenous RELN gene in HDMECs and reduce Reelin protein expression. Transfected HDMECs were subjected to cell‐based assays to assess changes in vascular endothelial cell behavior in response to RELN silencing. We assessed vascular endothelial cell adhesion on extracellular matrices that are required during coronary vessel development. Vessel integrity was examined by assaying endothelial cell membrane permeability and resistance in RELN‐silenced cells. Also, we examined the effect of Reelin expression on cell viability.ResultsRELN‐silenced HDMECs exhibited a >90% reduction in Reelin expression in comparison to non‐targeting, negative control HDMECs. This significant Reelin reduction altered vascular endothelial cell adhesion to certain extracellular matrices. These RELN‐silenced HDMECs exhibited a significant decrease in baseline cell membrane permeability and concomitant changes in cell membrane resistance. Furthermore, we observed changes in cell morphology in RELN‐silenced cells versus control cells. Preliminary data suggests that loss of RELN expression affects vascular endothelial cell morphology and expression of components within the Reelin signaling pathway.ConclusionsBased on this evidence, we conclude that Reelin influences adhesion, morphology, membrane permeability and membrane resistance of vascular endothelial cells in order to regulate vessel wall integrity. These findings may provide key evidence as to how Reelin expression in nascent vascular endothelial cells impacts formation of the coronary vessels during mammalian cardiovascular development.Support or Funding InformationAHA 17AIREA3360773 and PCOM Center for Chronic Disorders of AgingThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

No abstract