Nanping Wang

MicroRNA-19a mediates the suppressive effect of laminar flow on cyclin D1 expression in human umbilical vein endothelial cells

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/cm2, 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.

ADAMTS-7 Mediates Vascular Smooth Muscle Cell Migration and Neointima Formation in Balloon-Injured Rat Arteries

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 &agr; 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.

Angiotensin II up-regulates soluble epoxide hydrolase in vascular endothelium in vitro and in vivo

Epoxyeicosatrienoic acids (EETs), as metabolites of arachidonic acid, may function as antihypertensive and antiatherosclerotic mediators for vasculature. EETs are degraded by soluble epoxide hydrolase (sEH). Pharmacological inhibition and genetic ablation of sEH have been shown to increase the level of EETs, and treating angiotensin II (Ang II)-infused hypertension rats with sEH-selective inhibitors increased the levels of EETs, with attendant decrease in systolic blood pressure. To elucidate the mechanisms by which Ang II regulates sEH expression, we treated human umbilical vein endothelial cells (ECs) and bovine aortic ECs with Ang II and found increased sEH expression at both the mRNA and protein levels. Transient transfection assays showed that the activity of the human sEH promoter was increased in ECs in response to Ang II. Further analysis of the promoter region of the sEH gene demonstrated that treatment with Ang II, like overexpression of c-Jun/c-Fos, activates the sEH promoter through an AP-1-binding motif. The binding of c-Jun to the AP-1 site of the sEH promoter was confirmed by chromatin immunoprecipitation assays. In contrast, adenovirus overexpression of the dominant-negative mutant of c-Jun significantly attenuated the effects of Ang II on sEH induction. An elevated level of sEH was found in the aortic intima of both spontaneously hypertensive rats and Ang II-infused Wistar rats. Blocking Ang II binding to Ang II receptor 1 by losartan abolished the sEH induction. Thus, AP-1 activation is involved in the transcriptional up-regulation of sEH by Ang II in ECs, which may contribute to Ang II-induced hypertension.

Role of microRNA-23b in flow-regulation of Rb phosphorylation and endothelial cell growth

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 PS-induced 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.

Peroxisome proliferator‐activated receptor‐δ induces insulin‐induced gene‐1 and suppresses hepatic lipogenesis in obese diabetic mice

Primary nonalcoholic fatty liver disease is one of the most common forms of chronic liver diseases and is associated with insulin‐resistant states such as diabetes and obesity. Recent work has revealed potential implications of peroxisome proliferator‐activated receptor‐δ (PPARδ) in lipid homeostasis and insulin resistance. In this study, we examined the effect of PPARδ on sterol regulatory element‐binding protein‐1 (SREBP‐1), a pivotal transcription factor controlling lipogenesis in hepatocytes. Treatment with GW0742, the PPARδ agonist, or overexpression of PPARδ markedly reduced intracellular lipid accumulation. GW0742 and PPARδ overexpression in hepatocytes induced the expression of insulin‐induced gene‐1 (Insig‐1), an endoplasmic reticulum protein braking SREBP activation, at both the mRNA and the protein levels. PPARδ inhibited the proteolytic processing of SREBP‐1 into the mature active form, thereby suppressing the expression of the lipogenic genes fatty acid synthase, stearyl CoA desaturase‐1, and acetyl coenzyme A carboxylase. Our results revealed a direct binding of PPARδ to a noncanonical peroxisome proliferator responsive element motif upstream of the transcription initiation site of human Insig‐1. The disruption of this site diminished the induction of Insig‐1, which suggested that Insig‐1 is a direct PPARδ target gene in hepatocytes. Knockdown of endogenous Insig‐1 attenuated the suppressive effect of GW0742 on SREBP‐1 and its target genes, indicating PPARδ inhibited SREBP‐1 activation via induction of Insig‐1. Furthermore, overexpression of PPARδ by intravenous infection with the PPARδ adenovirus induced the expression of Insig‐1, suppressed SREBP‐1 activation, and, consequently, ameliorated hepatic steatosis in obese db/db mice. Conclusion: Our study reveals a novel mechanism by which PPARδ regulates lipogenesis, suggesting potential therapeutic applications of PPARδ modulators in obesity and type 2 diabetes, as well as related steatotic liver diseases. (HEPATOLOGY 2008.)

Suppression of Pro-inflammatory Adhesion Molecules by PPAR-δ in Human Vascular Endothelial Cells

Objective—Endothelial activation is implicated in atherogenesis and diabetes. The role of peroxisome proliferator-activated receptor-&dgr; (PPAR-&dgr;) in endothelial activation remains poorly understood. In this study, we investigated the anti-inflammatory effect of PPAR-&dgr; and the mechanism involved. Methods and Results—In human umbilical vein endothelial cells (HUVECs), the synthetic PPAR-&dgr; ligands GW0742 and GW501516 significantly inhibited tumor necrosis factor (TNF)-α–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 endothelial-leukocyte adhesion. Activation of PPAR-&dgr; 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-&dgr; to the vascular cell adhesion molecule (VCAM)-1 promoter. Small interfering RNA reduced endogenous PPAR-&dgr; expression but potentiated the suppressive effect of GW0742 on EC activation, which suggests that the nonliganded PPAR-&dgr; may have an opposite effect. Conclusions—We have demonstrated that ligand activation of PPAR-&dgr; 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-&dgr; agonists may have a potential for treating inflammatory diseases such as atherosclerosis and diabetes.

Metformin Protects Endothelial Function in Diet-Induced Obese Mice by Inhibition of Endoplasmic Reticulum Stress Through 5′ Adenosine Monophosphate–Activated Protein Kinase–Peroxisome Proliferator–Activated Receptor δ Pathway

Objective—5′ Adenosine monophosphate–activated protein kinase (AMPK) interacts with peroxisome proliferator–activated receptor &dgr; (PPAR&dgr;) 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 PPAR&dgr; remains unknown. We aim to investigate whether PPAR&dgr; 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 PPAR&dgr; wild-type mice but not in diet-induced obese PPAR&dgr; knockout littermates. Metformin and PPAR&dgr; 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 (PPAR&dgr; 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/PPAR&dgr; pathway in obese diabetic mice.

c-Jun triggers apoptosis in human vascular endothelial cells.

In endothelial cells (ECs), the transcription factor c-Jun is induced by a variety of stimuli that perturb EC function. To extend our understanding of the role of c-Jun in EC physiology, we have directed overexpression of c-Jun in human umbilical vein ECs by using a tetracycline-regulated adenoviral expression system. In this study, we report a novel observation using this system. Specific expression of c-Jun is a sufficient trigger for ECs to undergo apoptosis, as demonstrated by a set of combined assays including an ELISA specific for histone-associated DNA fragmentation, DNA laddering, and TdT-mediated dUTP nick end labeling (TUNEL). Tetracycline can effectively shut off c-Jun overexpression and prevent EC apoptosis. Cleavage of poly(ADP-ribose) polymerase was also detected in ECs overexpressing c-Jun. Moreover, inhibitors of cysteine proteases blocked the apoptosis, suggesting a caspase-associated mechanism involved in proapoptotic effects of c-Jun. To gain further insight into the role of c-Jun as a pathophysiological regulator of EC death, TAM67, a dominant-negative mutant of c-Jun, was overexpressed in human umbilical vein ECs to abrogate endogenous c-Jun/activator protein-1 activation. H(2)O(2)-triggered apoptosis was largely attenuated in ECs overexpressing TAM67. Together, these results suggest that c-Jun, as a proapoptotic molecule, may play a role in mediating the cell death program in vascular endothelium.

Flow Activation of AMP-Activated Protein Kinase in Vascular Endothelium Leads to Krüppel-Like Factor 2 Expression

Objective—Vascular endothelial cells (ECs) confer atheroprotection at locations of the arterial tree where pulsatile laminar flow (PS) exists with a high shear stress and a large net forward direction. We investigated whether the PS-induced expression of the transcription factor Krüppel-Like Factor 2 (KLF2) in cultured ECs and its expression in the mouse aorta is regulated by AMP-activated protein kinase (AMPK). Methods and Results—AMPK inhibition by Compound C or siRNA had a significant blocking effect on the PS-induced KLF2 expression. The induction of KLF2 by PS led to the increase in eNOS and the suppression of ET-1, which could be reversed by KLF2 siRNA. In addition, PS induced the phosphorylation of ERK5 and MEF2 which are necessary for the KLF2 expression. These mechanotransduction events were abrogated by the blockade of AMPK. Furthermore, the phosphorylation levels of ERK5 and MEF2, as well as the expression of KLF2, were significantly reduced in the aorta of AMPKα2 knockout mice when compared with wild-type control mice. Conclusion—The flow-mediated AMPK activation is a newly defined KLF2 regulatory pathway in vascular endothelium that acts via ERK5/MEF2.

Homocysteine Upregulates Resistin Production From Adipocytes In Vivo and In Vitro

OBJECTIVE—Homocysteine (Hcy) is epidemiologically related to insulin resistance, which has been speculated to be a low-grade systemic inflammatory condition. Resistin acts as a critical mediator of insulin resistance associated with inflammatory conditions. We aimed to determine whether Hcy can induce insulin resistance by directly regulating the expression and secretion of resistin from adipose tissue. RESEARCH DESIGN AND METHODS—The effect of Hcy on the expression and secretion of resistin and insulin resistance was investigated using primary rat adipocytes and mice with hyperhomocysteinemia (HHcy). RESULTS—Hcy impaired glucose transport and, particularly, the insulin signaling pathway as shown by decreased insulin-stimulated tyrosine phosphorylation of insulin receptor and insulin receptor substrate (IRS)-1, increased serine phosphorylation of IRS-1, and inhibited Akt phosphorylation both in vitro and in vivo, and these impairments were accompanied by an increase in resistin expression. Compared with normal mice, HHcy mice with a clinically relevant level of plasma Hcy (19 μmol/l) showed significantly increased resistin production from adipose tissue (33.38 ± 3.08 vs. 19.27 ± 1.71 ng/ml, P < 0.01). Hcy (300–1000 μmol/l) also increased mRNA expression of resistin in primary rat adipocytes in a time- and concentration-dependent manner, with maximal induction at 24 h of approximately fourfold with 1,000 μmol/l. In addition, Hcy-induced resistin expression attenuated by treatment with reactive oxygen species (ROS) scavengers, protein kinase C (PKC), and nuclear factor (NF)-κB inhibitors implies a role in the process for ROS, PKC, and NF-κB. CONCLUSIONS—HHcy may promote insulin resistance through the induction of resistin expression and secretion from adipocytes via the activation of the ROS-PKC–NF-κB pathway.