Tomoko Kanome

Human Urotensin II Accelerates Foam Cell Formation in Human Monocyte-Derived Macrophages

Human urotensin II (U-II), the most potent vasoconstrictor peptide identified to date, and its receptor (UT) are involved in hypertension and atherosclerosis. Acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) converts intracellular free cholesterol into cholesterol ester (CE) for storage in lipid droplets and plays an important role in the formation of macrophage-derived foam cells in atherosclerotic lesions. We examined the effects of U-II on ACAT-1 expression and CE accumulation in human monocyte-derived macrophages. U-II increased ACAT activity in a concentration-dependent manner after 7 days in monocyte primary culture. Immunoblotting analysis showed that U-II at 25 nmol/L increased ACAT-1 protein expression level by 2.5-fold, which was completely abolished by anti–U-II antibody, selective UT receptor antagonists (urantide and 4-aminoquinoline), a G-protein inactivator (GDP-&bgr;-S), a c-Src protein tyrosine kinase inhibitor (PP2), a protein kinase C (PKC) inhibitor (rottlerin), a mitogen-activated protein kinase kinase (MEK) inhibitor (PD98059), or a Rho kinase (ROCK) inhibitor (Y27632). Northern blotting analysis indicated that among the 4 ACAT-1 mRNA transcripts (2.8-, 3.6-, 4.3-, and 7.0-kb), the 2.8- and 3.6-kb transcript levels were selectively upregulated by ≈1.7-fold by U-II (25 nmol/L). Further, U-II (25 nmol/L) significantly increased acetylated LDL (acetyl-LDL)–induced CE accumulation in monocyte-derived macrophages but not scavenger receptor class A (SR-A) function as assessed by endocytic uptake of [125I]acetyl-LDL. Our results suggest that U-II may play a novel role in the formation of macrophage-derived foam cells by upregulating ACAT-1 expression via the UT receptor/G-protein/c-Src/PKC/MEK and ROCK pathways but not by SR-A, thus contributing to the relatively rapid development of atherosclerosis in hypertension.

Impact of Salusin-α and -β on Human Macrophage Foam Cell Formation and Coronary Atherosclerosis

Background— Human salusins, related bioactive polypeptides with mitogenic effects on vascular smooth muscle cells and fibroblasts and roles in hemodynamic homeostasis, may be involved in the origin of coronary atherosclerosis. Macrophage foam cell formation, characterized by cholesterol ester accumulation, is modulated by scavenger receptor (cholesterol influx), acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1; storage cholesterol ester converted from free cholesterol), and ATP-binding cassette transporter A1 (cholesterol efflux). Methods and Results— Serum salusin-α levels were decreased in 173 patients with angiographically proven coronary artery disease compared with 40 patients with mild hypertension and 55 healthy volunteers (4.9±0.6 versus 15.4±1.1 and 20.7±1.5 pmol/L, respectively; P<0.0001). Immunoreactive salusin-α and -β were detected in human coronary atherosclerotic plaques, with dominance of salusin-β in vascular smooth muscle cells and fibroblasts. After 7 days in primary culture, acetylated low-density lipoprotein–induced cholesterol ester accumulation in human monocyte-derived macrophages was significantly decreased by salusin-α and increased by salusin-β. Salusin-α significantly reduced ACAT-1 expression in a concentration-dependent manner. In contrast, salusin-β significantly increased ACAT-1 expression by 2.1-fold, with a maximal effect at 0.6 nmol/L. These effects of salusins were abolished by G-protein, c-Src tyrosine kinase, protein kinase C, and mitogen-activated protein kinase kinase inhibitors. ACAT activity and ACAT-1 mRNA levels were also significantly decreased by salusin-α and increased by salusin-β; however, neither salusin-α nor salusin-β affected scavenger receptor A function assessed by [125I]acetylated low-density lipoprotein endocytosis or scavenger receptor class A and ATP-binding cassette transporter A1 expression. Conclusions— Our results indicate that the 2 salusin isoforms have opposite effects on foam cell formation in human monocyte-derived macrophages. Development of atherosclerosis may be accelerated by salusin-β and suppressed by salusin-α via ACAT-1 regulation.

Human Urotensin II as a Link between Hypertension and Coronary Artery Disease

Hypertension is a well-known risk factor for atherosclerosis, but the molecular mechanisms that link elevated blood pressure to the progression of atherosclerosis remain unclear. Human urotensin II (U-II), the most potent endogenous vasoconstrictor peptide identified to date, and its receptor (UT receptor) are involved in the etiology of essential hypertension. In patients with essential hypertension, U-II infused into the forearm brachial artery has been shown to induce vasoconstriction. Recent studies have demonstrated elevated plasma U-II concentrations in patients with essential hypertension, diabetes mellitus, atherosclerosis, and coronary artery disease. U-II is expressed in endothelial cells, macrophages, macrophage-derived foam cells, and myointimal and medial vascular smooth muscle cells (VSMCs) of atherosclerotic human coronary arteries. UT receptors are present in VSMCs of human coronary arteries, the thoracic aorta and cardiac myocytes. Lymphocytes are the most active producers of U-II, whereas monocytes and macrophages are the major cell types expressing UT receptors, with relatively little receptor expression in foam cells, lymphocytes, and platelets. U-II accelerates foam cell formation by up-regulation of acyl-coenzyme A:cholesterol acyltransferase-1 in human monocyte-derived macrophages. In human endothelial cells, U-II promotes cell proliferation and up-regulates type 1 collagen expression. U-II also activates nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and plasminogen activator inhibitor-1 in human VSMCs, and stimulates VSMC proliferation with synergistic effects observed when combined with oxidized low-density lipoprotein, lysophosphatidylcholine, reactive oxygen species or serotonin. These findings suggest that U-II plays key roles in accelerating the development of atherosclerosis, thereby leading to coronary artery disease.

Leptin modulates ACAT1 expression and cholesterol efflux from human macrophages

Leptin is an adipose tissue-derived hormone implicated in atherosclerosis and macrophage foam cell formation. The current study was conducted to examine the effect of leptin on cholesteryl ester accumulation in human monocytes/macrophages. Exogenously added leptin at 5 nM during differentiation of monocytes into macrophages for 7 days accelerated acetylated LDL (acetyl-LDL)-induced cholesteryl ester accumulation by 30-50%. Leptin did not affect endocytic uptake of acetyl-LDL; however, it increased ACAT activity 1.8-fold and ACAT-1 protein expression 1.9-fold. Among the four ACAT-1 mRNA transcripts, two shorter transcripts (2.8 and 3.6 kb) were upregulated approximately 1.7-fold upon leptin treatment. The enhanced expression of ACAT-1 protein by leptin was suppressed by inhibitors of Janus-activated kinase2 (JAK2) and phosphatidylinositol 3-kinase (PI3K). HDL-mediated cholesterol efflux was suppressed by leptin, which was canceled by K-604, an ACAT-1 inhibitor. Expression of long form of leptin receptor was upregulated during monocytic differentiation into macrophages and sustained after differentiation. Thus, the results suggest that leptin accelerates cholesteryl ester accumulation in human monocyte-derived macrophages by increasing ACAT-1 expression via JAK2 and PI3K, thereby suppressing cholesterol efflux.

Inhibitors of Acyl-Coenzyme A: Cholesterol Acyltransferase

Acyl-coenzyme A: cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes the formation of cholesterol esters from cholesterol and fatty acyl-coenzyme A. Animal experiments showed that ACAT inhibitors reduce plasma cholesterol levels by suppressing absorption of dietary cholesterol and by suppressing the assembly and secretion of apolipoprotein B-containing lipoproteins such as very low density lipoprotein in liver and chylomicron in intestine. Moreover, ACAT inhibitors were shown to prevent formation of macrophage-derived foam cells in the arterial walls. However, a recent double-blind, placebo-controlled, randomized trial of a potent ACAT inhibitor, avasimibe, failed to show significant beneficial effects on coronary atherosclerosis assessed by intravascular ultrasound. For clinical application of ACAT inhibitors, development of more potent compounds and improvements of the methods to evaluate their clinical efficacy are strongly needed.

Human urotensin-II potentiates the mitogenic effect of mildly oxidized low-density lipoprotein on vascular smooth muscle cells: comparison with other vasoactive agents and hydrogen peroxide.

Human urotensin-II (U-II) is the most potent vasoactive peptide identified to date, and may be involved in hypertension and atherosclerosis. We investigated the effects of the interactions between U-II or other vasoactive agents and mildly oxidized low-density lipoprotein (mox-LDL) or hydrogen peroxide (H2O2) on the induction of vascular smooth muscle cell (VSMC) proliferation. Growth-arrested rabbit VSMCs were incubated with vasoactive agents (U-II, endothelin-1, angiotensin-II, serotonin, or thromboxane-A2) in the presence or absence of mox-LDL or H2O2. [3H]Thymidine incorporation into DNA was measured as an index of VSMC proliferation. On interaction with mox-LDL or H2O2, U-II induced the greatest increase in [3H]thymidine incorporation among these vasoactive agents. A low concentration of U-II (10 nmol/l) enhanced the potential mitogenic effect of low concentrations of mox-LDL (120 to 337%) and H2O2 (177 to 226%). U-II at 50 nmol/l showed the maximal mitogenic effect (161%), which was abolished by G protein inactivator (GDP-β-S), c-Src tyrosine kinase inhibitor (radicicol), protein kinase C (PKC) inhibitor (Ro31-8220), extracellular signal–regulated kinase (ERK) kinase inhibitor (PD98059), or Rho kinase inhibitor (Y27632). Mox-LDL at 5 μg/ml showed the maximal mitogenic effect (211%), which was inhibited by free radical scavenger (catalase), intracellular and extracellular antioxidants (N-acetylcysteine and probucol), nicotinamide adenine dinucleotide phosphate oxidase inhibitor (diphenylene iodonium), or c-Jun N-terminal kinase (JNK) inhibitor (SP600125). These results suggested that U-II acts in synergy with mox-LDL in inducing VSMC DNA synthesis at the highest rate among these vasoactive agents. Activation of the G protein/c-Src/PKC/ERK and Rho kinase pathways by U-II together with the redox-sensitive JNK pathway by mox-LDL may explain the synergistic interaction between these agents.

Angiotensin II upregulates acyl-CoA:cholesterol acyltransferase-1 via the angiotensin II Type 1 receptor in human monocyte-macrophages.

Angiotensin II (Ang II) is known to accelerate the progression of macrophage-driven atherosclerotic lesions. Acyl-CoA:cholesterol acyltransferase-1 (ACAT1) converts intracellular free cholesterol into cholesterol ester (CE) for storage in lipid droplets, and promotes foam cell formation in atherosclerotic lesions. The present study explored the effect of Ang II on ACAT1 expression as a molecular mechanism of foam cell formation in primary cultured human monocyte-macrophages. Ang II significantly increased ACAT1 protein expression in a time- or concentration-dependent manner. Application of an Ang II type 1 (AT1) receptor agonist (L162313), but not an Ang II type 2 (AT2) receptor agonist (CGP42112A), mimicked the effects of Ang II treatment in inducing ACAT1 protein expression. ACAT activity and ACAT1 mRNA levels were also significantly increased by Ang II. Two-fold increases in ACAT1 protein expression and ACAT activity with Ang II treatment were completely inhibited by AT1 receptor antagonists (candesartan, [Sar1,IIe8]-Ang II), but not by an AT2 receptor antagonist (PD123319). Treatment with a G-protein inactivator (GDP-β-S), a c-Src tyrosine kinase inhibitor (PP2), a protein kinase C (PKC) inhibitor (rottlerin), or a mitogen activated protein kinase (MAPK) kinase inhibitor (PD98059) significantly reduced Ang II–induced ACAT1 protein expression. Macrophage foam cell formation assessed using acetylated low-density lipoprotein (LDL)–induced CE accumulation was significantly enhanced by Ang II, which was completely inhibited by treatment with candesartan. These results suggested that Ang II enhances foam cell formation by upregulating ACAT1 expression predominantly through the actions of AT1 receptor via the G protein/c-Src/PKC/MAPK pathway in human monocyte-macrophages.

Abstract 1639: The novel c-MET inhibitor, ARQ 197, shows additive growrh-inhibitory effect with erlotinib through enhanced degradation of c-MET protein via ubiquitin/proteasome pathway

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnBackgroundnnc-MET, a receptor of hepatocyte growth factor (HGF), is known to be overexpressed in a variety of human cancers. Recently several c-MET inhibitors are being developed as cancer chemotherapy against this compelling molecular target. ARQ 197 is a new small molecule c-MET inhibitor. Since ARQ 197 shows efficient anti-cancer activity without many severe adverse events in Phase I clinical trials, it is expected to be a useful anticancer agent for clinical treatment. This drug is known to bind to c-MET at neighbor area of its ATP binding site and it does not work as an ATP mimic. In this study, we investigated the molecular mechanism of inhibitory effect on c-MET by ARQ 197 in a non-small cell lung cancer cell (NSCLC) line.nnMaterials and MethodnnWe established a c-MET-overexpressed NSCLC line, PC-9/MET, by long term exposure of PC-9 cells to EGFR-tyrosine kinase inhibitor. The cytotoxicity of ARQ 197 was measured by MTT assay. The expression of c-MET protein and mRNA were detected by Western blotting analysis and real time RT-PCR method, respectively. In a c-MET degradation analysis, bortezomib was used for inhibition of proteasome.nnResultsnnARQ 197 is equally cytotoxic to parental PC-9 and EGFRTKI-resistant PC-9/MET cell line with GI50 values around 200 nM (?). ARQ 197 showed an additive growth- inhibitory effect with erlotinib in PC-9/MET cells as assessed by an Isobologram analysis. In ARQ 197-treated PC-9/MET cells, a clear decrease of phospho c-MET protein as well as phospho AKT and ERK proteins was observed, however, ARQ 197 also induced a down-regulation of c-MET protein in the cells without any effect on c-MET mRNA level. This ARQ 197 induced degradation was reversed by a concomitant treatment with a proteasome inhibitor, bortezomib in the cells. However, bortezomib did not affect cytotoxicity of ARQ 197 in the same cells.nnConclusionnnThese data suggest that ARQ 197 may inhibit c-MET through downregulation of this protein in addition to inhibition of its kinase activity. Furthermore, it may enhance c-MET degradation by the ubiquitin/proteasome pathway. These c-MET inhibitory activities by ARQ 197 should be important for the additive combined effect with erlotinib in PC-9/MET cells.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1639.

Relationship Between Hypertension and Atherosclerosis: From a Viewpoint of the Most Potent Vasoconstrictor Human Urotensin II

Human urotensin II (U-II), the most potent vasoconstrictor peptide identified to date, and its receptor (UT) are involved in etiology of hypertension. In hypertensive patients, U-II induces vasoconstriction in forearm brachial artery infusion studies. Recent studies demonstrated elevated plasma U-II concentrations in patients with hypertension, diabetes mellitus, atherosclerosis, and coronary artery disease. U-II is expressed in endothelial cells, macrophages, macrophagederived foam cells, and myointimal and medial vascular smooth muscle cells (VSMCs) of atherosclerotic human coronary arteries. UT receptors are present in VSMCs of human coronary arteries, thoracic aorta and cardiac myocytes. Lymphocytes are the most active producers of U-II, whereas monocytes and macrophages are the major cell types expressing UT receptors, with relatively little receptor expression in foam cells, lymphocytes, and platelets. U-II accelerates foam cell formation by up-regulation of acyl-coenzyme A:cholesterol acyltransferase-1 in human monocyte-derived macrophages, and stimulates cell growth and up-regulates type 1 collagen expression in human endothelial cells. U-II also activates NADPH oxidase and plasminogen activator inhibitor-1 in human VSMCs, and stimulates VSMC proliferation with synergistic effects observed when combined with oxidized LDL, reactive oxygen species and serotonin. These findings suggest that U-II plays key roles in accelerating the development of atherosclerosis, and hence coronary artery disease.

Abstract 699: HSP70 specifically binds to 15 bp deletion mutant EGFR and modulates sensitivity to EGFR-TKI

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnn[Purpose] Non small lung cancer (NSCLC) cells that expressed mutant EGFR are more sensitive to EGFR-tyrosine kinase inhibitors (EGFR-TKIs) than that expressed wild type EGFR. Recent clinical trials revealed that more than 70% NSCLC patients that expressed mutant EGFR showed sensitivity to EGFR-TKIs, such as gefitinib and erlotinib. To elucidate the mechanism of this hypersensitivity, we explored the difference of EGFR-binding proteins between wild type EGFR and 15 bp deletion mutant EGFR using respective stable transfectants.nn[Methods] Wild type EGFR and a 15 bp deletion mutant EGFR plasmids were transfected into HEK293 cells. The stable transfectant cells were established, and were designated 293_pEGFR and 293_pΔ15, respectively. Cells were incubated in a medium with/without 10 ng/ml of TGFα for 1 h and lysed. Cell lysate was precleared by centrifugation at 15,000 rpm for 15 min. The supernatant was mixed with a polyclonal EGFR antibody for 1 h and EGFR was immunoprecipitated by Protein A-Sepharose. After adequate washing, coprecipitated proteins that bound to EGFR were eluted and separated by 2D-PAGE (Immobiline DryStrip (pH3-10 NL, 7 cm) and 10% SDS-PAGE). Proteins were visualized by silver staining and identified by LC-MS/MS. HSP70 siRNA was transfected into the cells by lipofection method. Sensitivity to gefitinib was measured by MTT assay. EGFR binding affinity to gefitinib was measured using [14C] gefitinib.nn[Results and Discussion] We detected several EGFR binding proteins. Among these proteins, one candidate (lesser binding to wild type EGFR than the mutant EGFR) was identified as HSP 70 by LC-MS/MS. The total amount of HSP70 protein was not different between 293_pEGFR and293_pΔ15 cells. A specific binding of this protein to the mutant EGFR was confirmed by Western blotting analysis. This binding was not modulated by EGFR activation. Knockdown of HSP 70 protein by a specific siRNA enhanced gefitinib sensitivity in the mutant EGFR transfectant.nnThese results suggest that HSP 70 may decrease sensitivity to EGFR-TKIs in the cells that expressed 15 bp deletion mutant EGFR. There is a possibility that HSP70 overexpression might be a newly resistant mechanism to EGFR-TKIs in NSCLC patients that expressed mutant EGFR.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 699. doi:10.1158/1538-7445.AM2011-699