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/

Objective-Atherosclerosis is an inflammatory disease in which macrophage activation and lipid loading play a crucial role. In this study, we investigated expression and function of the NR4A nuclear receptor family, comprising Nur77 (NR4A1, TR3), Nurr1 (NR4A2), and NOR-1 (NR4A3) in human macrophages. Methods and Results-Nur77, Nurr1, and NOR-1 are expressed in early and advanced human atherosclerotic lesion macrophages primarily in areas of plaque activation/progression as detected by in situ-hybridization and immunohistochemistry. Protein expression localizes to the nucleus. Primary and THP-1 macrophages transiently express NR4A-factors in response to lipopolysaccharide and tumor necrosis factor ␣. Lentiviral overexpression of Nur77, Nurr1, or NOR-1 reduces expression and production of interleukin (IL)-1␤ and IL-6 proinflammatory cytokines and IL-8, macrophage inflammatory protein-1␣ and -1␤ and monocyte chemoattractant protein-1 chemokines. In addition, NR4A-factors reduce oxidized-low-density lipoprotein uptake, consistent with downregulation of scavenger receptor-A, CD36, and CD11b macrophage marker genes. Knockdown of Nur77 or NOR-1 with gene-specific lentiviral short-hairpin RNAs resulted in enhanced cytokine and chemokine synthesis, increased lipid loading, and augmented CD11b expression, demonstrating endogenous NR4A-factors to inhibit macrophage activation, foam-cell formation, and differentiation. A therosclerosis is a chronic inflammatory disease involving deregulation of both the immune system and lipid metabolism. 1,2 Macrophages, imperative in the innate immune system, are involved in the initiation, progression, and rupture of atherosclerotic lesions as well as in the initiation of smooth muscle cell (SMC)-rich pathologies like restenosis. 3,4 At the onset of atherosclerosis, monocytes are locally recruited to the arterial vessel wall, where these cells differentiate into macrophages. These intimal macrophages ingest modified lipid particles and become lipid-laden foam cells that form a so-called fatty streak. In advanced atherosclerotic lesions, macrophages are localized primarily around a central lipid core and at the shoulder region of the plaque. At the latter site, which is known to be prone to rupture, these cells may be involved in destabilization of the lesion. 5 Throughout the progression of atherosclerosis, macrophages produce proinflammatory cytokines, chemokines, growth factors, and matrix-degrading enzymes and are consequently crucial in the chronic inflammatory process in the diseased vessel wall. 6,7 Detailed knowledge on the molecular mechanisms involved in the inflammatory and metabolic processes in macrophages is essential to develop novel drug therapies against atherosclerosis. We hypothesized that NR4A nuclear receptors are key regulatory factors involved in modulation of these specific processes in macrophages. Conclusion-NR4A-factorsThe NR4A nuclear hormone receptors were first described as early response transcription factors expressed on stimulation by growth factors. 8 -...

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Transcription factor complexes bind to regulatory sequences of genes, providing a system of individual expression regulation. Targets of distinct transcription factors usually map throughout the genome, without clustering. Nevertheless, highly and weakly expressed genes do cluster in separate chromosomal domains with an average size of 80-90 genes. We therefore asked whether, besides transcription factors, an additional level of gene expression regulation exists that acts on chromosomal domains. Here we show that identical green fluorescent protein (GFP) reporter constructs integrated at 90 different chromosomal positions obtain expression levels that correspond to the activity of the domains of integration. These domains are up to 80 genes long and can exert an eightfold effect on the expression levels of integrated genes. 3D-FISH shows that active domains of integration have a more open chromatin structure than integration domains with weak activity. These results reveal a novel domain-wide regulatory mechanism that, together with transcription factors, exerts a dual control over gene transcription.