Endothelial cells (ECs) respond to changes in mechanical forces, leading to the modulation of signaling networks and cell function; an example is the inhibition of EC proliferation by steady laminar flow. MicroRNAs (miRs) are short noncoding 20-22 nucleotide RNAs that negatively regulate the expression of target genes at the posttranscriptional level. This study demonstrates that miRs are involved in the flow regulation of gene expression in ECs. With the use of microRNA chip array, we found that laminar shear stress (12 dyn/cm 2 , 12 h) regulated the EC expression of many miRs, including miR-19a. We further showed that stable transfection of miR-19a significantly decreased the expression of a reporter gene controlled by a conserved 3′-untranslated region of the cyclinD1 gene and also the protein level of cyclin D1, leading to an arrest of cell cycle at G1/S transition. Laminar flow suppressed cyclin D1 protein level, and this suppressive effect was diminished when the endogenous miR-19a was inhibited. In conclusion, we demonstrated that miR-19a plays an important role in the flow regulation of cyclin D1 expression. These results revealed a mechanism by which mechanical forces modulate endothelial gene expression.

Chemokines are important mediators in immune responses and inflammatory processes. Calcitonin gene-related peptide (CGRP) is produced in dorsal root ganglion (DRG) neurons. In this study, CGRP radioimmunoassay was used to investigate whether the chemokines CCL2 and CXCL1 could trigger CGRP release from cultured DRG neurons of neonatal rats and, if so, which cellular signaling pathway was involved. The results showed that CCL2 and CXCL1 ( approximately 5-100 ng/ml) evoked CGRP release and intracellular calcium elevation in a pertussis toxin (PTX)-sensitive manner. The CGRP release by CCL2 and CXCL1 was significantly inhibited by EGTA, omega-conotoxin GVIA (an N-type calcium channel blocker), thapsigargin, and ryanodine. Pretreatment of DRG neurons for 30 min with the inhibitors of phospholipase C (PLC) and protein kinase C (PKC) but not mitogen-activated protein kinases (MAPKs) significantly reduced CCL2- or CXCL1-induced CGRP release and intracellular calcium elevation. Intraplantar injection of CCL2 or CXCL1 produced hyperalgesia to thermal and mechanical stimulation in rats. These data suggest that CCL2 and CXCL1 can stimulate CGRP release and intracellular calcium elevation in DRG neurons. PLC-, PKC-, and calcium-induced calcium release from ryanodine-sensitive calcium stores signaling pathways are involved in CCL2- and CXCL1-induced CGRP release from primary nociceptive neurons, in which chemokines produce painful effects via direct actions on chemokine receptors expressed by nociceptive neurons.

Objective-Endothelial activation is implicated in atherogenesis and diabetes. The role of peroxisome proliferator-activated receptor-␦ (PPAR-␦) in endothelial activation remains poorly understood. In this study, we investigated the anti-inflammatory effect of PPAR-␦ and the mechanism involved. Methods and Results-In human umbilical vein endothelial cells (HUVECs), the synthetic PPAR-␦ ligands GW0742 and GW501516 significantly inhibited tumor necrosis factor (TNF)-␣-induced expression of vascular cell adhesion molecule-1 and E-selectin (assayed by real-time RT-PCR and Northern blotting), as well as the ensuing endothelialleukocyte adhesion. Activation of PPAR-␦ upregulated the expression of antioxidant genes superoxide dismutase 1, catalase, and thioredoxin and decreased reactive oxygen species production in ECs. Chromatin immunoprecipitation assays showed that GW0742 switched the association of BCL-6, a transcription repressor, from PPAR-␦ to the vascular cell adhesion molecule (VCAM)-1 promoter. Small interfering RNA reduced endogenous PPAR-␦ expression but potentiated the suppressive effect of GW0742 on EC activation, which suggests that the nonliganded PPAR-␦ may have an opposite effect. Conclusions-We have demonstrated that ligand activation of PPAR-␦ in ECs has a potent antiinflammatory effect, probably via a binary mechanism involving the induction of antioxidative genes and the release of nuclear corepressors. PPAR-␦ agonists may have a potential for treating inflammatory diseases such as atherosclerosis and diabetes.