Spatial and temporal regulation of gap junction connexin43 in vascular endothelial cells exposed to controlled disturbed flows in vitro

Hemodynamic regulation of the endothelial gap junction protein connexin43 ( Cx43) was studied in a model of controlled disturbed flows in vitro. Cx43 m RNA, protein expression, and intercellular communication were mapped to spa tial variations in fluid forces. Hemodynamic features of atherosclerotic le sion-prone regions of the, vasculature (flow separation and recirculation) were created for periods of 5, 16, and 30 h, with laminar shear stresses ra nging between 0 and 13.5 dynes/cm(2). Within I h, endothelial Cx43 mRNA exp ression was increased in all cells when compared with no-flow controls, Wit h highest levels (up to 6- to 8-fold) expressed in regions of flow recircul ation corresponding to high shear stress gradients. At 16 hi Cx43 mRNA expr ession remained elevated in regions of flow disturbance, whereas in areas o f fully developed, undisturbed laminar flaw? Cx43 expression returned to co ntrol levels. In all flow regions, typical punctate Cx43 immunofluorescence at cell borders was disrupted by 5 h, After 30 h of flow, disruption of ga p junctions persisted in cells subjected to flow separation and recirculati on, whereas regions of undisturbed flow were substantially restored to norm al. These expression differences were reflected in sustained inhibition of intercellular communication (dye transfer) throughout the zone of disturbed flow (84.2 and 68.4%, inhibition at 5 and 30 h, respectively); in contrast , communication was fully reestablished by 30 h in cells exposed to undistu rbed flow. Up-regulation of Cx43 transcripts, sustained disorganization of Cx43 protein, and impaired communication suggest that shear stress gradient s in regions of disturbed flow regulate intercellular communication through the expression and function of Cx43.