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.

Combination effect between bortezomib and tumor necrosis factor alpha on gefitinib-resistant non-small cell lung cancer cell lines.

Tumor cells that have acquired resistance to gefitinib may complicate the future treatment of patients with non-small cell lung cancer (NSCLC). To investigate the mechanisms of acquired resistance, an acquired gefitinib-resistant cell line, PC-9/ZD2001, has been established using a gefitinib-sensitive NSCLC cell line, PC-9. PC-9/ZD2001 showed collateral sensitivity to tumor necrosis factor (TNF)-alpha. Bortezomib is a proteasome inhibitor and enhances TNF-alpha-induced cell death. These observations suggest that the combination of bortezomib and TNF-alpha might have effects against gefitinib-resistant cells. To verify this hypothesis, a combination effect between these drugs was examined using MTT assay and immunoblotting. This combination showed synergistic cytotoxic effect in NSCLC cell lines with either acquired or intrinsic gefitinib resistance. However, this combination effect was not observed in gefitinib-sensitive cells. On the other hand, bortezomib inhibited TNF-alpha-induced IkappaB degradation in all cell lines. From these observations, it is concluded that the combination of bortezomib and TNF-alpha could be used to overcome gefitinib-resistance.

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 (AT(1)) receptor agonist (L162313), but not an Ang II type 2 (AT(2)) 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 AT(1) receptor antagonists (candesartan, [Sar(1),Ile(8)]-Ang II), but not by an AT(2) receptor antagonist (PD123319). Treatment with a G-protein inactivator (GDP-beta-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 AT(1) receptor via the G protein/c-Src/PKC/MAPK pathway in human monocyte-macrophages.

Impact of salusin-alpha and -beta 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-alpha 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-alpha and -beta were detected in human coronary atherosclerotic plaques, with dominance of salusin-beta 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-alpha and increased by salusin-beta. Salusin-alpha significantly reduced ACAT-1 expression in a concentration-dependent manner. In contrast, salusin-beta 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-alpha and increased by salusin-beta; however, neither salusin-alpha nor salusin-beta 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-beta and suppressed by salusin-alpha 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.

Serotonin acts as an up-regulator of acyl-coenzyme A:cholesterol acyltransferase-1 in human monocyte-macrophages.

Acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) converts intracellular free cholesterol into cholesterol ester for storage in lipid droplets and plays an important role in the formation of macrophage-derived foam cells in atherosclerotic lesions. Serotonin (5-HT), a potent vasoconstrictor that is released from activated platelets, increases uptake of oxidized low-density lipoprotein (LDL) by macrophages, leading to foam cell formation, and contributes to the development of atherosclerotic plaque. However, it is not yet known whether 5-HT affects ACAT-1 expression in human monocyte-macrophages as the molecular mechanism of enhanced foam cell formation by 5-HT remains unclear. We examined the effects of 5-HT on ACAT-1 expression during differentiation of cultured human monocytes into macrophages. Expression of ACAT-1 protein but not 5-HT2A receptor increased in a time-dependent manner. 5-HT increased ACAT activity in a concentration-dependent manner after 7 days in primary monocyte culture. Immunoblotting analysis showed that 5-HT at 10 microM increased ACAT-1 protein expression level by two-fold, and this effect was abolished completely by a 5-HT2A receptor antagonist (sarpogrelate), its major metabolite (M-1), a G protein inactivator (GDP-beta-S), a protein kinase C (PKC) inhibitor (rottlerin), a Src family inhibitor (PP2), or a mitogen-activated protein kinase (MAPK) kinase inhibitor (PD98059). Northern blotting analysis indicated that among the four ACAT-1 mRNA transcripts (2.8-, 3.6-, 4.3-, and 7.0-kb), the levels of the 2.8- and 3.6-kb transcripts were selectively up-regulated by approximately 1.7-fold by 5-HT (10 microM). The results of the present study suggested that 5-HT may play a crucial role in macrophage-derived foam cell formation by up-regulating ACAT-1 expression via the 5-HT2A receptor/G protein/c-Src/PKC/MAPK pathway, contributing to the progression of atherosclerotic plaque.

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-beta-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 microg/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.

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-beta-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 approximately 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 [(125)I]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.

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.