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.

The migration of vascular smooth muscle cells (VSMCs) plays an essential role during the development of atherosclerosis and restenosis. Extensive studies have implicated the importance of extracellular matrix (ECM)-degrading proteinases in VSMC migration. A recently described family of proteinases, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTs), is capable of degrading vascular ECM proteins. Here, we sought to determine whether ADAMTS-7 is involved in VSMC migration and neointima formation in response to vascular injury. ADAMTS-7 protein accumulated preferentially in neointima of the carotid artery wall after balloon injury. In primary VSMCs, ADAMTS-7 level was enhanced by the proinflammatory cytokine tumor necrosis factor α and growth factor platelet-derived growth factor-BB. ADAMTS-7 overexpression greatly accelerated and small interfering RNA knockdown markedly retarded VSMC migration/invasion in vitro. In addition, luminal delivery of ADAMTS-7 adenovirus to carotid arteries exacerbated intimal thickening nearly sixfold 7 days after injury. Conversely, perivascular administration of ADAMTS-7 small interfering RNA but not scramble small interfering RNA to injured arteries attenuated intimal thickening by 50% at 14 days after injury. Furthermore, ADAMTS-7 mediated degradation of the vascular ECM cartilage oligomeric matrix protein (COMP) in injured vessels. Replenishing COMP circumvented the promigratory effect of ADAMTS-7 on VSMCs. Enforced expression of COMP significantly suppressed VSMC migration and neointima formation postinjury, which indicates that ADAMTS-7 facilitated intimal hyperplasia through degradation of inhibitory matrix protein COMP. ADAMTS-7 may therefore serve as a novel therapeutic target for atherosclerosis and postangioplasty restenosis.

Peroxisome proliferator-activated receptor-γ (PPAR-γ) is a ligand-activated nuclear receptor that has an essential role in adipogenesis and glucose homeostasis. PPAR-γ is expressed in vascular tissues including endothelial cells (ECs). PPAR-γ activity can be regulated by many pathophysiological and pharmacological agonists. However, the role of PPAR-γ activation in ECs remains unclear. In this study, we examined the effect of the constitutive activation of PPAR-γ on the phenotypic modulation of ECs. Adenovirus-mediated expression of a constitutively active mutant of PPAR-γ resulted in significant ligand-independent activation of PPAR-γ and specific induction of the PPAR-γ target genes. However, PPAR-γ activation significantly suppressed the expression of vascular adhesion molecules in ECs and the ensuing leukocyte recruitment. Furthermore, constitutive activation of PPAR-γ resulted in simultaneous repression of AP-1 and NF-κB activity, which suggests that PPAR-γ may reduce pro-inflammatory phenotypes via, at least in part, suppression of the AP-1 and NF-κB pathways. Therefore, using a gain-of-function approach, our study provides novel evidence showing that constitutive activation of PPAR-γ is sufficient to prevent ECs from converting into a pro-inflammatory phenotype. These results also suggest that, in addition to pharmacological agonists, the genetic modification of the PPAR-γ activity in ECs may be a potential approach for therapeutic intervention in various inflammatory disorders.

Objective—: 5′ Adenosine monophosphate–activated protein kinase (AMPK) interacts with peroxisome proliferator–activated receptor δ (PPARδ) to induce gene expression synergistically, whereas the activation of AMPK inhibits endoplasmic reticulum (ER) stress. Whether the vascular benefits of antidiabetic drug metformin (AMPK activator) in diabetes mellitus and obesity is mediated by PPARδ remains unknown. We aim to investigate whether PPARδ is crucial for metformin in ameliorating ER stress and endothelial dysfunction induced by high-fat diet. Approach and Results—: Acetylcholine-induced endothelium-dependent relaxation in aortae was measured on wire myograph. ER stress markers were determined by Western blotting. Superoxide production in mouse aortae and NO generation in mouse aortic endothelial cells were assessed by fluorescence imaging. Endothelium-dependent relaxation was impaired and ER stress markers and superoxide level were elevated in aortae from high-fat diet–induced obese mice compared with lean mice. These effects of high-fat diet were reversed by oral treatment with metformin in diet-induced obese PPARδ wild-type mice but not in diet-induced obese PPARδ knockout littermates. Metformin and PPARδ agonist GW1516 reversed tunicamycin (ER stress inducer)-induced ER stress, oxidative stress, and impairment of endothelium-dependent relaxation in mouse aortae as well as NO production in mouse aortic endothelial cells. Effects of metformin were abolished by cotreatment of GSK0660 (PPARδ antagonist), whereas effects of GW1516 were unaffected by compound C (AMPK inhibitor). Conclusions—: Metformin restores endothelial function through inhibiting ER stress and oxidative stress and increasing NO bioavailability on activation of AMPK/PPARδ pathway in obese diabetic mice.