The Yorkie homologues YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif, also known as WWTR1), effectors of the Hippo pathway, have been identified as mediators for mechanical stimuli. However, the role of YAP/TAZ in haemodynamics-induced mechanotransduction and pathogenesis of atherosclerosis remains unclear. Here we show that endothelial YAP/TAZ activity is regulated by different patterns of blood flow, and YAP/TAZ inhibition suppresses inflammation and retards atherogenesis. Atheroprone-disturbed flow increases whereas atheroprotective unidirectional shear stress inhibits YAP/TAZ activity. Unidirectional shear stress activates integrin and promotes integrin-Gα interaction, leading to RhoA inhibition and YAP phosphorylation and suppression. YAP/TAZ inhibition suppresses JNK signalling and downregulates pro-inflammatory genes expression, thereby reducing monocyte attachment and infiltration. In vivo endothelial-specific YAP overexpression exacerbates, while CRISPR/Cas9-mediated Yap knockdown in endothelium retards, plaque formation in ApoE mice. We also show several existing anti-atherosclerotic agents such as statins inhibit YAP/TAZ transactivation. On the other hand, simvastatin fails to suppress constitutively active YAP/TAZ-induced pro-inflammatory gene expression in endothelial cells, indicating that YAP/TAZ inhibition could contribute to the anti-inflammatory effect of simvastatin. Furthermore, activation of integrin by oral administration of MnCl reduces plaque formation. Taken together, our results indicate that integrin-Gα-RhoA-YAP pathway holds promise as a novel drug target against atherosclerosis.

Abstract-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 ␣ 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. Key Words: metalloproteinase Ⅲ vascular smooth muscle cell migration Ⅲ neointima formation Ⅲ extracellular matrix M edia-to-intima migration of vascular smooth muscle cells (VSMCs) is pivotal to intimal thickening in atherosclerosis, restenosis after coronary angioplasty, and late failure of vein grafting. 1 Normally VSMCs are quiescent and are surrounded by and embedded in an extracellular matrix (ECM) scaffold that acts as a barrier to VSMC migration. ECM degradation and remodeling require the activation of extracellular proteases, which in turn facilitate VSMC migration. 2 Previous studies have emphasized potential roles for the matrix metalloproteinases MMP-2, MMP-9, and MT1-MMP; the serine proteinases plasminogen activator and plasminogen; and the cysteine proteinases cathepsins K, L, and S during matrix remodeling and VSMC migration. 3 However, the identity of the matrix-degrading proteinases during pathological vascular remodeling in vivo has remained the subject of speculation.The recently identified metalloproteinase family of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) also degrade ECM. First identified in 1997, ADAMTS already showed strong biological relevance. 4 For example, ADAMTS...

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

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.

endothelial cells ͉ arachidonic acid ͉ AP-1 ͉ promoter ͉ hypertension A rachidonic acid (AA) derived from membrane phospholipids plays a key role in vascular inflammatory and/or antiinflammatory responses. AA can be converted to eicosanoids by three major enzymatic pathways, namely, cyclooxygenase, lipoxygenase, and CYP 450 epoxygenase. Exerting autocrine effects on vascular endothelial cells (ECs), four epoxyeicosatrienoic acids (EETs) regioisomers 5,6-, 8,9-, 11,12-, and 14,15-EET are the major metabolites generated by CYP 450 epoxygenase (1). EETs can be released by ECs to act as paracrine mediators on neighboring cells such as vascular smooth muscle cells (VSMCs) (2). EETs exert membrane potential-independent effects and modulate several signaling cascades that affect EC proliferation and angiogenesis. EETs also function as endothelium-derived hyperpolarizing factors (3). By increasing intracellular Ca 2ϩ concentration, EETs activate large conductance Ca 2ϩ -activated K ϩ channel (BK Ca ) in the smooth muscle. The activation of BK Ca then causes hyperpolarization of VSMCs and subsequent vasodilation, which lowers the blood pressure (4). As well, EETs inhibit cytokine-induced inflammatory responses in ECs (5, 6). Treating ECs with 11,12-EET or overexpression of CYP2J2 attenuated the TNF␣-, IL-1␣-, and LPSinduced expression of adhesion molecules in ECs, thus decreasing leukocyte adhesion to the vascular wall (7).Epoxide hydrolases (EHs) convert epoxides to the corresponding diols. Under physiological conditions, EETs can be enzymatically hydrolysed to dihydroxyeicosatrienoic acids (DHETs) by EHs (1). Two major EHs in the ␣/␤ hydrolase family exist in mammalian cells: soluble EH (sEH), which primarily presents in the cytosol and peroxisomes, and microsomal EH, which binds to the intracellular membranes (8). Highly expressed in the liver, kidney, intestine, and vasculature, sEH is the main enzyme that converts 5,8,11,14,8,11,14, respectively. The mammalian sEH is a homodimer, and each subunit contains a C-and an N-terminal domain. The active site is located in the C-terminal domain in which the residues Asp-333, Asp-495, and His-523 form the catalytic triad (9). DHETs are much more polar than EETs and are generally considered as biologically inactive products of EETs. However, their roles are not fully understood.Angiotensin II (Ang II), a potent vessel constrictor, elevates blood pressure in various animal models. i.p. injection of sEHselective inhibitors to Ang II-infused hypertensive rats greatly increased the level of EETs and lowered systolic blood pressure (10). Thus, augmentation of EET levels with enhanced production by CYP450s or decreased hydrolysis by sEH seems to control blood pressure in vivo. In line with this hypothesis, recent studies demonstrated that the selective sEH inhibitor Ncyclohexyl-N-dodecyl urea reversed the hypertensive phenotype in the spontaneously hypertensive rat (SHR) (11).We have previously shown that laminar shear stress, an atheroprotective flow, decreased the expression of sE...

Summary Recent evidence suggests that the intestine may play a direct facilitative role in reverse cholesterol transport (RCT), independent of hepatobiliary secretion. In order to understand the non-biliary pathway for RCT we created both genetic and surgical models of biliary cholesterol insufficiency. To genetically inhibit biliary cholesterol secretion we generated mice in which Niemann-Pick C1-Like 1 (NPC1L1) was overexpressed in the liver. Compared to controls, NPC1L1Liver-Tg mice exhibit a > 90% decrease in biliary cholesterol secretion, yet mass fecal sterol loss and macrophage RCT is normal. To surgically inhibit biliary emptying into the intestine, we have established an acute biliary diversion model. Strikingly, macrophage RCT persists in mice surgically lacking the ability to secrete bile into the intestine. Collectively, these studies demonstrate that mass fecal sterol loss and macrophage RCT can proceed in the absence of biliary sterol secretion, challenging the obligate role of bile in RCT.

The retinoblastoma susceptibility gene (RB) encodes a phosphoprotein of 110 kd (pp110RB) that forms specific complexes with SV40 T antigen and the transforming proteins of several other DNA tumor viruses. Interaction with RB is thought to contribute to transformation by these viruses as demonstrated by genetic analyses. To help understand the function of these interactions, the regions of RB that are involved in binding to T have been mapped. An in vitro protein synthesis system capable of producing full‐length RB protein has been developed to facilitate the mapping study. A 5‐ to 10‐fold increase in translational efficiency in the reticulocyte lysate was obtained when the 5′ non‐coding region of RB mRNA was replaced with that of beta‐globin mRNA or a plant viral RNA, alfalfa mosaic virus (AMV) RNA4. A series of mutated RB polypeptides produced from this system were assayed for T binding. Two non‐contiguous regions of the RB protein, amino acid residues 394‐571 and 649‐773, were found to be necessary for binding to T: mutations in either region abolished T‐RB complex formation. These results are consistent with the finding that, in all the cases analyzed so far, mutated RB proteins in human tumor cells also failed to bind to T antigen due to deletions including at least one of the two required regions. Thus the regions of RB defined in vitro as necessary for interaction with T might be physiologically relevant as well, and might play a fundamental role in normal RB protein function.

MicroRNAs (miRs) can regulate many cellular functions, but their roles in regulating responses of vascular endothelial cells (ECs) to mechanical stimuli remain unexplored. We hypothesize that the physiological responses of ECs are regulated by not only mRNA and protein signaling networks, but also expression of the corresponding miRs. EC growth arrest induced by pulsatile shear (PS) flow is an important feature for flow regulation of ECs. miR profiling showed that 21 miRs are differentially expressed (8 up-and 13 downregulated) in response to 24-h PS as compared to static condition (ST). The mRNA expression profile indicates EC growth arrest under 24-h PS. Analysis of differentially expressed miRs yielded 68 predicted mRNA targets that overlapped with results of microarray mRNA profiling. Functional analysis of miR profile indicates that the cell cycle network is highly regulated. The upregulation of miR-23b and miR-27b was found to correlate with the PS-induced EC growth arrest. Inhibition of miR-23b using antagomir-23b oligonucleotide (AM23b) reversed the PSinduced E2F1 reduction and retinoblastoma (Rb) hypophosphorylation and attenuated the PS-induced G1/G0 arrest. Antagomir AM27b regulated E2F1 expression, but did not affect Rb and growth arrest. Our findings indicate that PS suppresses EC proliferation through the regulation of miR-23b and provide insights into the role of miRs in mechanotransduction.cell cycle | shear | bioinformatics | gene regulation | mechanotransduction H emodynamic forces, e.g., stretch and shear stress, act constantly on the vascular endothelial cells (ECs) to modulate EC signaling, gene expression, and physiological functions (1). Atherosclerotic lesions in the arterial tree are found mainly at branch points, where blood flow is disturbed with a limited forwarding direction, but are generally spared at the straight parts of the arterial tree, where the flow is laminar with a large forwarding direction (2). Exposure of ECs to 24 h of steady laminar shear flow at 12 dyn/cm 2 (approximating the hemodynamic force in straight parts of arteries) leads to antiproliferative (3) and antiinflammatory (4) responses. In contrast, ECs exposed to disturbed flow, mimicking the hemodynamic force at branch points, exhibit opposite responses (5, 6). The laminar shear-induced EC growth arrest involves the expression of CDK inhibitors (e.g., p21 cip , p27 kip ), tumor suppressor p53, and retinoblastoma (Rb) hypophosphorylation (3, 7). Whereas there is considerable knowledge on mechanotransduction at protein and mRNA levels, there is little information on the role of microRNAs (miRs) in this process.miRs are small noncoding RNAs (∼21-25 nucleotides) that regulate gene expression by binding to target mRNAs to cause their degradation or translational repression (8). It is estimated that miRs regulate ∼30% of human protein-coding genes. More than 800 miRs have been identified in the human genome and registered in the Sanger miRBase. These small RNAs provide a powerful mechanism for posttranscriptional cont...