Chronic infusion of salusin-alpha and -beta exerts opposite effects on atherosclerotic lesion development in apolipoprotein E-deficient mice.

OBJECTIVE: Human salusin-alpha and -beta are two-related peptides processed from the same precursor, preprosalusin. Our previous in vitro studies have shown that human macrophage foam cell formation is stimulated by salusin-beta but suppressed by salusin-alpha. Thus we investigated the effects of salusin-alpha and -beta on atherosclerotic plaque formation in vivo in apolipoprotein E-deficient (ApoE-/-) mice. METHODS: Saline (vehicle), salusin-alpha or -beta (0.6 nmol/kg/h) was continuously infused through osmotic mini-pumps into 13-week-old ApoE-/- mice for 8 weeks. Aortic atherosclerosis, oxidized LDL-induced cholesterol ester accumulation (foam cell formation), and its related gene expression in exudate peritoneal macrophages were determined. RESULTS: After 4-week infusion of salusin-beta, atherosclerotic lesions were 2.6 times greater than vehicle controls, which paralleled 1.9-fold increase in foam cell formation and up-regulation of scavenger receptors (CD36, scavenger receptor class A) and acyl-CoA: cholesterol acyltransferase-1 (ACAT1). In contrast, salusin-alpha decreased serum total cholesterol levels by 15% and foam cell formation by 68% associated with ACAT1 down-regulation. After 8-week infusion of salusin-alpha, atherosclerotic lesions were significantly suppressed by 54% compared with vehicle controls. CONCLUSIONS: Our study provided the first evidence that salusin-beta accelerates the development of atherosclerotic lesions associated with up-regulation of scavenger receptors and ACAT1 in ApoE-/- mice. Whilst, salusin-alpha exerts anti-atherosclerotic effects by suppressing serum total cholesterol levels and ACAT1 expression.

Preventive effects of heregulin-beta1 on macrophage foam cell formation and atherosclerosis.

RATIONALE: Human heregulins, neuregulin-1 type I polypeptides that activate proliferation, differentiation, and survival of glial cells, neurons, and myocytes, are expressed in macrophage foam cells within human coronary atherosclerotic lesions. Macrophage foam cell formation, characterized by cholesterol ester accumulation, is modulated by scavenger receptor class A (SR-A), acyl-coenzyme A:cholesterol acyltransferase (ACAT)1, and ATP-binding cassette transporter (ABC)A1. OBJECTIVE: The present study clarified the roles of heregulins in macrophage foam cell formation and atherosclerosis. METHODS AND RESULTS: Plasma heregulin-beta(1) levels were significantly decreased in 31 patients with acute coronary syndrome and 33 patients with effort angina pectoris compared with 34 patients with mild hypertension and 40 healthy volunteers (1.3+/-0.3, 2.0+/-0.4 versus 7.6+/-1.4, 8.2+/-1.2 ng/mL; P<0.01). Among all patients with acute coronary syndrome and effort angina pectoris, plasma heregulin-beta(1) levels were further decreased in accordance with the severity of coronary artery lesions. Expression of heregulin-beta(1) was observed at trace levels in intracoronary atherothrombosis obtained by aspiration thrombectomy from acute coronary syndrome patients. Heregulin-beta(1), but not heregulin-alpha, significantly reduced acetylated low-density lipoprotein-induced cholesterol ester accumulation in primary cultured human monocyte-derived macrophages by reducing SR-A and ACAT1 expression and by increasing ABCA1 expression at both mRNA and protein levels. Heregulin-beta(1) significantly decreased endocytic uptake of [(125)I]acetylated low-density lipoprotein and ACAT activity, and increased cholesterol efflux to apolipoprotein (Apo)A-I from human macrophages. Chronic infusion of heregulin-beta(1) into ApoE(-/-) mice significantly suppressed the development of atherosclerotic lesions. CONCLUSIONS: This study provided the first evidence that heregulin-beta(1) inhibits atherogenesis and suppresses macrophage foam cell formation via SR-A and ACAT1 downregulation and ABCA1 upregulation.

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.

Human urotensin II promotes hypertension and atherosclerotic cardiovascular diseases.

Human urotensin II (U-II), the most potent vasoconstrictor undecapeptide identified to date, and its receptor (UT) are involved in the pathogenesis of systemic and pulmonary hypertension. Here, we review recent advances in our understanding of the pathophysiology of U-II with particular reference to its role in atherosclerotic cardiovascular diseases. Single-nucleotide polymorphisms of U-II gene (S89N) are associated with onset of essential hypertension, type II diabetes mellitus, and insulin resistance in the Asian population. Plasma U-II levels are elevated in patients with vascular endothelial dysfunction-related diseases such as essential hypertension, diabetes mellitus, atherosclerosis, ischemic heart disease, and heart failure. Chronic infusion of U-II enhances atherosclerotic lesions in the aorta in apolipoprotein E-knockout mice. In human atherosclerotic plaques from the aorta and coronary and carotid arteries, U-II is expressed at high levels in endothelial cells (ECs) and lymphocytes, whereas UT is expressed at high levels in vascular smooth muscle cells (VSMCs), ECs, monocytes, and macrophages. U-II stimulates vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression in human ECs as chemoattractant for monocytes, and accelerates foam cell formation by up-regulation of acyl-coenzyme A:cholesterol acyltransferase-1 in human monocyte-derived macrophages. U-II produces reactive oxygen species (ROS) via nicotinamide adenine dinucleotide phosphate oxidase activation in human VSMCs, and stimulates VSMC proliferation with synergistic effects when combined with ROS, oxidized LDL, and serotonin. Clinical studies demonstrated increased plasma U-II levels in accordance with the severity of carotid atherosclerosis in patients with essential hypertension and that of coronary artery lesions in patients with ischemic heart disease. Here, we summarize the key roles of U-II in progression of hypertension and atherosclerotic cardiovascular diseases.

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.

Chronic urotensin II infusion enhances macrophage foam cell formation and atherosclerosis in apolipoprotein E-knockout mice.

OBJECTIVE: Our recent studies have indicated that urotensin II, the most potent vasoconstrictor peptide identified to date, potentiates human macrophage foam cell formation and vascular smooth muscle cell proliferation, and its levels are increased in the plasma of hypertensive patients with carotid atherosclerotic plaques. In the present study, we investigated the enhancing effect of urotensin II on atherosclerosis in apolipoprotein E-knockout mice and its suppression by 4-aminoquinoline, an urotensin II receptor-selective antagonist. METHODS: Urotensin II, urotensin II + 4-aminoquinoline, or vehicle was infused for 4 weeks through an osmotic mini-pump into 9-week-old apolipoprotein E-knockout mice on a high-fat diet. Aortic atherosclerosis and foam cell formation in exudate peritoneal macrophages were examined. RESULTS: Atherosclerotic lesions as well as plasma levels of urotensin II, reactive oxygen species, and oxidized low-density lipoprotein and oxidized low-density lipoprotein-induced foam cell formation were significantly greater in urotensin II-infused mice than vehicle-infused controls. Western blotting analysis showed increased expression of scavenger receptors (CD36 and scavenger receptor class A) and acyl-CoA:cholesterol acyltransferase-1 in these macrophages. Increases in these parameters were significantly reduced by addition of 4-aminoquinoline. In apolipoprotein E-knockout mice even without urotensin II infusion, the treatment with 4-aminoquinoline for 8 weeks significantly prevented the development of atherosclerotic lesions. CONCLUSION: Our results provide the first evidence that increased plasma urotensin II level stimulates oxidized low-density lipoprotein and reactive oxygen species production and macrophage foam cell formation via increased expression of CD36, scavenger receptor class A, and acyl-CoA:cholesterol acyltransferase-1, contributing to the development of atherosclerosis in apolipoprotein E-deficient mice. Urotensin II receptor antagonism may be a promising therapeutic strategy against atherosclerosis.

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.

Ndrg4 enhances NGF-induced ERK activation uncoupled with Elk-1 activation.

Ndrg4 is expressed predominantly in the early postnatal rat brain and may be related to neural cell differentiation. PC12 cell lines stably expressing increased levels of Ndrg4 protein display enhanced NGF-induced phosphorylation of MEK and ERK. In contrast, the Ndrg4-C2-overexpressed PC12 cell lines showed attenuated NGF-promoted phosphorylation of Elk-1, which is a nuclear target of ERK. A reporter assay also indicated that Ndrg4-C2 suppresses Elk-1-mediated transcriptional activation and SRE reporter expression. The suppressive effect of Ndrg4-C2 on NGF-induced activation of Elk-1 was abolished by colchicine but not by cytochalasin D, suggesting that microtubules are involved in the reduced activation of Elk-1 by Ndrg4. Ndrg4 may play a role in supporting the activation of ERK and its target proteins needed for neuronal differentiation and in reducing the activation of Elk-1 implicated in cell growth.

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.

Genomic organization, expression, and comparative analysis of noncoding region of the rat Ndrg4 gene.

Rat Ndrg4 is a member of the NDRG gene family and has been suggested to relate to brain development. The structure of the rat Ndrg4 gene was studied to understand the mechanism for the expression of multiple forms of Ndrg4 protein, which were revealed in the brain. Subcloning and DNA sequencing analysis of a bacterial artificial chromosome (BAC) clone, together with analysis of a transcriptional start site by a cap-site hunting, indicated that the Ndrg4 gene spans about 39 kilobases (kb) and consists of 19 exons, in which the first and second exons were first found in rat. An alternative promoter usage at different transcriptional start sites may produce three types of messages, Ndrg4-A, Ndrg4-B, and Ndrg4-C, and there is a variant that lacks exon 18 for each type of transcript. Thereby, Ndrg4-A1, Ndrg4-A2, Ndrg4-B1, Ndrg4-B2, Ndrg4-C1, and Ndrg4-C2 were identified to be expressed. These six variants might explain the heterogeneity of the Ndrg4 protein in the brain. The variants without exon 18 were revealed in the embryonic and early postnatal brains while those with exon 18 were detected in the maturing and adult brains. Radiation hybrid mapping suggests that the rat Ndrg4 gene is located on chromosome 19 at 90.6 centirays (cR) from the top. Comparison of the noncoding sequence of the rat Ndrg4 gene to those of the orthologous mouse and human genes suggests that the AP-1 binding site is a candidate regulatory element.

Inhibition of neurite outgrowth by reduced level of NDRG4 protein in antisense transfected PC12 cells.

NDRG4, a member of the new NDRG gene family, was originally cloned as a gene that was expressed predominantly in the early postnatal rat brain. To determine whether the NDRG4 protein contributes to differentiation of neural cells, the effect of lowering the cellular NDRG4 protein level on the nerve growth factor (NGF)-induced neurite formations and transcription factor activations in PC12 cells was examined. An antisense construct of rat NDRG4 cDNA was made and transfected to PC12 cells, which constitutively express a basal level of the NDRG4 protein. Of the stably transfected antisense cell clones that expressed exogenous NDRG4 antisense RNA, six clones showed reduced levels of the NDRG4 protein, but unexpectedly two clones showed quite higher levels of NDRG4 protein than the control cells. The clones having decreased levels of the NDRG4 protein extended shorter neurites than control cells in response to NGF or dibutyryl cAMP. In contrast, the NDRG4 protein-highly expressing clones did not show suppressed neurite outgrowth induced by NGF. NGF-mediated activation of the transcription factor AP-1 was found to be suppressed in the NDRG4 protein-diminished clone and enhanced in the NDRG4 protein-upregulated clone as compared with those in the control cells. These results suggest that NDRG4 plays a role in neurite outgrowth and has an influence on an NGF-stimulated AP-1 activation by an undefined mechanism in PC12 cells.

Molecular characterization of NDRG4/Bdm1 protein isoforms that are differentially regulated during rat brain development.

We previously reported the identification of a novel gene, Bdm1/NDRG4, that was expressed predominantly in the postnatal rat brain and might possibly play a role in this process. We describe here the characterization of a NDRG4 protein in a developing and maturing rat brain. Antibody raised against glutathione S-transferase (GST)-NDRG4 fusion protein recognized four protein species of 38, 39, 41, and 45 kDa on Western blotting of proteins from differently staged rat brains. The 38-kDa form was revealed after birth, and the amount of this species peaked on postnatal day 15. The 39-kDa form became detectable after postnatal week 6. The 41-kDa form appeared late in embryogenesis, increased by postnatal day 15, and disappeared at postnatal week 6. The 45-kDa form was abundant during the late embryonic period and slightly decreased after birth. Subcellular fractionation of cerebra indicated that the NDRG4 protein was distributed mainly in the mitochondria and endoplasmic reticulum (ER). Detergent solubility assays and protease susceptibility demonstrated that in the ER NDRG4 protein is membrane-associated and luminally oriented. The 45-kDa isoform was induced during NGF-mediated neuronal differentiation of PC12 cells, but not by tunicamycin which causes ER stress. Differential expressions of NDRG4 protein isoforms may be a mechanism for modifying the NDRG4 function and for the formation of a functioning nervous system.

Effect of lignins and their precursors on nitric oxide, citrulline and asparagine production by mouse macrophage-like Raw 264.7 cells.

Lignins, tannins and flavonoids are commonly found polyphenols. Among these polyphenols, lignins, polymers of phenylpropenoids complexed with polysaccharides, were the least cytotoxic and most potently stimulated the production of nitric oxide (NO), citrulline and asparagine by mouse macrophage-like Raw 264.7 cells. The maximum production of these substances reached the level attained by lipopolysaccharide (LPS). However, epigallocatechin gallate, phenylpropenoid monomers (ferulic acid, caffeic acid) and gallic acid (component unit of tannin) were inactive. These data suggest that the macrophage-stimulation activity of polyphenols depends, at least in part, on their molecular weight or structural configuration. There was a positive relationship between the extent of asparagine production and that of NO or citrulline. Western blot analysis demonstrated that both lignins and LPS elevated the cellular level of asparagine synthetase. The present study suggests the possible link between the stimulated asparagine production and macrophage activation.