The endothelium expresses a large repertoire of genes under apparent transcriptional control of biomechanical forces, many of which are neither cell-type nor flow specific. We set out to identify genes that are uniquely flow responsive in human vascular endothelial cells. Transcriptional profiling using commercial DNA microarrays identified 12 of 18 000 genes that were modulated at least 5-fold after 24 hours of steady laminar flow (25 dyne/ cm 2 ). After a 7-day exposure to unidirectional pulsatile flow (19 ؎ 12 dyne/cm 2 ), only 3 of 12 remained elevated at least 5-fold. A custom microarray of ϳ300 vascular cell-related gene fragments was constructed, and expression analysis revealed that many flow-induced genes are also induced by at least one of the following agents: tumor necrosis factor-␣ (TNF-␣), interleukin-1␤ (IL-1␤), transforming growth factor-␤, vascular endothelial growth factor, or thrombin, indicating a more general role in adaptive or stress responses. Most flow-induced genes were also induced by TNF-␣ but not IL-1␤, suggesting the involvement of reactive oxygen species. A limited panel of genes that are unique for flow-exposed cultures was identified, including lung Krü ppellike factor (LKLF/KLF2) and cytochrome P450 1B1 (CYP1B1). In marked contrast, both these genes were substantially repressed by TNF-␣. LKLF but not CYP1B1 mRNA was detected exclusively in the vascular endothelium of healthy human aorta by in situ hybridization and appeared to be flow regulated. To date LKLF is the first endothelial transcription factor that is uniquely induced by flow and might therefore be at the molecular basis of the physiological healthy, flow-exposed state of the endothelial cell.

Lung Krüppel-like factor (LKLF/KLF2) is an endothelial transcription factor that is crucially involved in murine vasculogenesis and is specifically regulated by flow in vitro. We now show a relation to local flow variations in the adult human vasculature: decreased LKLF expression was noted at the aorta bifurcations to the iliac and carotid arteries, coinciding with neointima formation. The direct involvement of shear stress in the in vivo expression of LKLF was determined independently by in situ hybridization and laser microbeam microdissection/reverse transcriptase-polymerase chain reaction in a murine carotid artery collar model, in which a 4-to 30-fold induction of LKLF occurred at the high-shear sites. Dissection of the biomechanics of LKLF regulation in vitro demonstrated that steady flow and pulsatile flow induced basal LKLF expression 15-and 36-fold at shear stresses greater than ϳ5 dyne/cm 2 , whereas cyclic stretch had no effect. The focal development of atherosclerosis has been linked to the local variations in blood flow that are observed near the irregular blood vessel geometries of bifurcations and bends. 1,2 Continuous exposure of endothelial cells to flow in vivo generates a tangential force, shear stress, across their apical surfaces. A large number of studies support the hypothesized antiatherosclerotic effect of shear stress on the endothelium, and are mainly based on the ability of shear stress to modulate endothelial gene expression. 3 Throughout the recent years, a collection of shear stress-responsive endothelial genes has been established. 4 -8 Usually no clear distinction is made between genes induced by prolonged shear and those induced by short-term shear (Ͻ24 hours), although the latter class typically represents a general stress response also observed with turbulent flow types and seems more related to endothelial dysfunction. Based on the rationale that only genes induced by prolonged shear would represent the healthy transcriptome, we previously identified a limited number of genes that are still highly induced after exposing human umbilical vein endothelial cells (HUVECs) to flow for 7 days, but which are not induced by various other (inflammatory) stimuli. 6 The expression of one of those genes, the transcription factor lung Krü ppel-like factor (LKLF/