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A principal function of endothelial cells is the formation of a barrier between the blood and tissues.  This barrier arises from the physical connections at cell-cell junctions, which includes cytoskeletal tight junction and adherens junction proteins. Methods that alter barrier function must therefore affect these cell-cell connections. The blood brain barrier (BBB) represents perhaps the most selective endothelial barrier, which arises from endothelial cell interactions with astrocytes and pericytes. Even in non-central nervous system (CNS) endothelial cells, barrier properties can be enhanced, mimicking the BBB, through induction of intercellular junctions, by either direct co-culture with astrocytes, supplementation with astrocyte conditioned medium (ACM) and/or pharmacologic enhancement of cAMP. To understand how cell-cell junctions change during endothelial barrier enhancement, we examined the effects of ACM and/or cAMP donors added to standard media on human umbilical vein endothelial cells (HUVEC). HUVEC cultured with cAMP-elevating agents had the most enhanced barrier function as measured by Electric Cell-substrate Impedance Sensing (ECIS®), a real-time, label-free, impedance based method of studying cell barrier properties. However, subtle differences in actin and cell-cell junction proteins were seen across all four culture conditions. cAMP-elevating agents also triggered the redistribution of ZO-1 and VE-cadherin to cell-cell junctions, and intensified the actin microfilament network at the cell cortex.  Using a VE-cadherin FRET-force sensor, we observed a decrease in VE-cadherin force in HUVEC cultured with ACM with cAMP donors. Our data indicate cAMP elevation induces both junctional strengthening and reduced VE-cadherin forces. Additionally, treatment with an inhibitor of formin, which reduced actin stress fibers, enhanced barrier function. These data suggest that barrier function is modulated both through the trafficking of proteins to cell-cell junctions, and through the modulation and a relaxation of mechanical force through adherens junctions as intercellular junctional complexes become established.