In the arterial circulation, regions of disturbed flow (DF), which are characterized by flow separation and transient vortices, are susceptible to atherogenesis, whereas regions of undisturbed laminar flow (UF) appear protected. Coordinated regulation of gene expression by endothelial cells (EC) may result in differing regional phenotypes that either favor or inhibit atherogenesis. Linearly amplified RNA from freshly isolated EC of DF (inner aortic arch) and UF (descending thoracic aorta) regions of normal adult pigs was used to profile differential gene expression reflecting the steady state in vivo. By using human cDNA arrays, Ϸ2,000 putatively differentially expressed genes were identified through false-discovery-rate statistical methods. A sampling of these genes was validated by quantitative realtime PCR and͞or immunostaining en face. Biological pathway analysis revealed that in DF there was up-regulation of several broad-acting inflammatory cytokines and receptors, in addition to elements of the NF-B system, which is consistent with a proinflammatory phenotype. However, the NF-B complex was predominantly cytoplasmic (inactive) in both regions, and no significant differences were observed in the expression of key adhesion molecules for inflammatory cells associated with early atherogenesis. Furthermore, there was no histological evidence of inflammation. Protective profiles were observed in DF regions, notably an enhanced antioxidative gene expression. This study provides a public database of regional EC gene expression in a normal animal, implicates hemodynamics as a contributory mechanism to athero-susceptibility, and reveals the coexistence of pro-and antiatherosclerotic transcript profiles in susceptible regions. The introduction of additional risk factors may shift this balance to favor lesion development.

Hemodynamic regulation of the endothelial gap junction protein connexin43 (Cx43) was studied in a model of controlled disturbed f lows in vitro. Cx43 mRNA, protein expression, and intercellular communication were mapped to spatial variations in f luid forces. Hemodynamic features of atherosclerotic lesion-prone regions of the vasculature (f low separation and recirculation) were created for periods of 5, 16, and 30 h, with laminar shear stresses ranging between 0 and 13.5 dynes͞cm 2 . Within 5 h, endothelial Cx43 mRNA expression was increased in all cells when compared with no-f low controls, with highest levels (up to 6-to 8-fold) expressed in regions of f low recirculation corresponding to high shear stress gradients. At 16 h, Cx43 mRNA expression remained elevated in regions of f low disturbance, whereas in areas of fully developed, undisturbed laminar f low, Cx43 expression returned to control levels. In all f low regions, typical punctate Cx43 immunof luorescence at cell borders was disrupted by 5 h. After 30 h of f low, disruption of gap junctions persisted in cells subjected to f low separation and recirculation, whereas regions of undisturbed f low were substantially restored to normal. These expression differences were ref lected in sustained inhibition of intercellular communication (dye transfer) throughout the zone of disturbed f low (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 undisturbed f low. Up-regulation of Cx43 transcripts, sustained disorganization of Cx43 protein, and impaired communication suggest that shear stress gradients in regions of disturbed f low regulate intercellular communication through the expression and function of Cx43.Endothelial cell communication throughout the confluent monolayer as well as with circulating leukocytes and underlying smooth muscle cells plays an important role in vessel wall homeostasis and atherosclerosis (1). The localization of atherosclerotic lesions coincides with regions of disturbed blood flows where endothelial cells exhibit an altered phenotype characterized by polygonal cell shape, enhanced mitotic index, and a striking impairment of endothelial-dependent vasoresponsiveness, characteristics that have been shown to be associated with alterations in cell-cell communication (2-4). In vitro, fluid dynamic studies demonstrate a close relationship between flow characteristics and altered endothelial morphology and function (5-7). Taken together, in vivo and in vitro studies suggest a link between endothelial intercellular communication and hemodynamic conditions that may be relevant to focal vulnerability to atherosclerosis.Intercellular communication occurs by a combination of humoral exchange (e.g., cytokines) and by direct transfer of signals between neighboring cells through transmembrane gap junctional assemblies composed of connexin (Cx) proteins (8). Endothelial cells express Cx40, 37, and 43, of which the most prominent connexin in vitro is Cx43 (...