Background Upregulated by atheroprotective flow, the transcription factor Krüppel-like factor 2 (KLF2) is crucial for maintaining endothelial function. MicroRNAs (miRNAs) are non-coding small RNAs that regulate gene expression at the post-transcriptional level. We examined the role of miRNAs, particularly miR-92a, in the atheroprotective flow-regulated KLF2. Methods and Results Dicer knockdown increased the level of KLF2 mRNA in human umbilical vein endothelial cells (HUVECs), suggesting that KLF2 is regulated by miRNA. In silico analysis predicted that miR-92a could bind to the 3’ untranslated region (3’UTR) of KLF2 mRNA. Overexpression of miR-92a precursor (pre-92a) decreased the expression of KLF2 and the KLF2-regulated endothelial nitric oxide synthase (eNOS) and thrombomodulin (TM) at mRNA and protein levels. A complementary finding is that miR-92a inhibitor (anti-92a) increased the mRNA and protein expression of KLF2, eNOS, and TM. Subsequent studies revealed that atheroprotective laminar flow downregulated the level of miR-92a to induce KLF2 and the level of this flow-induced KLF2 was reduced by pre-92a. Furthermore, miR-92a level was lower in HUVECs exposed to the atheroprotective pulsatile shear flow (PS) than under atheroprone oscillatory shear flow. Anti-Ago1/2 immunopreciptation coupled with RT-PCR revealed that PS decreased the functional targeting of miR-92a/KLF2 mRNA in HUVECs. Consistent with these findings, mouse carotid arteries receiving pre-92a exhibited impaired vasodilatory response to flow. Conclusions Atheroprotective flow patterns decrease the level of miR-92a, which in turn increases KLF2 expression to maintain endothelial homeostasis.

Rationale Endothelial microRNA-126 (miR-126) modulates vascular development and angiogenesis. However, its role in the regulation of smooth muscle cell (SMC) function is unknown. Objective To elucidate the role of miR-126 secreted by endothelial cells (ECs) in regulating SMC turnover in vitro and in vivo, as well as the effects of shear stress (SS) on the regulation. Methods and Results Co-culture of SMCs with ECs or treatment of SMCs with conditioned media from static EC monoculture (EC-CM) increased SMC miR-126 level and SMC turnover; these effects were abolished by inhibition of endothelial miR-126 and by the application of laminar SS (LSS) to ECs. SMC miR-126 did not increase when treated with EC-CM from ECs subjected to inhibition of miR biogenesis, nor with CM from sheared ECs. Depletion of extracellular/secreted vesicles in EC-CM did not affect the increase of SMC miR-126 by EC-CM. Biotinylated miR-126 or FLAG-tagged Ago2 transfected into ECs were detected in the co-cultured or EC-CM treated SMCs, indicating a direct EC-to-SMC transmission of miR-126 and Ago2. Endothelial miR-126 represses FOXO3, BCL2, and IRS1 mRNAs in the co-cultured SMCs, suggesting the functional roles of the transmitted miR-126. Systemic depletion of miR-126 in mice inhibited neointimal lesion formation of carotid arteries induced by cessation of blood flow. Administration of EC-CM or miR-126 mitigated the inhibitory effect. Conclusions Endothelial miR-126 acts as a key intercellular mediator to increase SMC turnover, and its release is reduced by atheroprotective LSS.

This study was designed to elucidate the mechanism underlying the inhibition of endothelial cell growth by laminar shear stress. Tumor suppressor gene p53 was increased in bovine aortic endothelial cells subjected to 24 h of laminar shear stress at 3 dynes (1 dyne ‫؍‬ 10 N)͞cm 2 or higher, but not at 1.5 dynes͞cm 2 . One of the mechanisms of the shear-induced increase in p53 is its stabilization after phosphorylation by c-Jun N-terminal kinase. To investigate the consequence of the shear-induced p53 response, we found that prolonged laminar shear stress caused increases of the growth arrest proteins GADD45 (growth arrest and DNA damage inducible protein 45) and p21 cip1 , as well as a decrease in phosphorylation of the retinoblastoma gene product. Our results suggest that prolonged laminar shear stress causes a sustained p53 activation, which induces the up-regulation of GADD45 and p21 cip1 . The resulting inhibition of cyclin-dependent kinase and hypophosphorylation of retinoblastoma protein lead to endothelial cell cycle arrest. This inhibition of endothelial cell proliferation by laminar shear stress may serve an important homeostatic function by preventing atherogenesis in the straight part of the arterial tree that is constantly subjected to high levels of laminar shearing. H emodynamic forces regulate the structure and function of the blood vessel wall (1, 2). Vascular endothelial cells (ECs), located at the interface between the circulating blood and the blood vessel, are exposed to shear stresses resulting from the tangential forces exerted by the flowing fluid on the vessel wall. The magnitude and pattern of the shear stress acting on ECs depend on blood flow, blood viscosity, and the vascular geometry, which varies with the location in the vascular tree. In regions of the vascular tree that have predilection for atherosclerotic lesions (e.g., branch points of large to medium arteries), the complex flow pattern is associated with low shear stresses that exhibit large spatial variations. In contrast, in the straight parts of the arterial tree, which are generally spared from atherosclerosis, blood flow is more laminar, and the high level of shear stress shows little spatial variations. Previous in vitro and in vivo findings have shown that ECs respond to shear stress in a magnitude-and flow pattern-dependent manner (3-5). ECs subjected to a long duration of laminar shear stress at the relatively high levels seen in the straight part of the arterial tree [i.e., on the order of 10-20 dynes (1 dyne ϭ 10 N)͞cm 2 ] have been found to have a lower rate of DNA synthesis than that under static condition (6). This shear-induced reduction of DNA synthesis, which indicates a decrease in cell proliferation, is not seen in ECs subjected to low shear stresses at 1-5 dynes͞cm 2 (6-8). The molecular mechanisms by which EC growth is regulated by the high level of sustained laminar shear stress seen in the lesion-resistant part of the arterial tree have not yet been clearly established. The elucidation of these mechanisms...