Contributions of an Extracellular Matrix Glycoprotein to Coronary Vessel Formation

Abstract

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.