Hydrogen peroxide-inducible clone 5 (Hic-5) as a potential therapeutic target for vascular and other disorders.

Hydrogen peroxide-inducible clone-5 (Hic-5) is a focal adhesion scaffold protein primarily expressed in vascular and visceral smooth muscle cells. We recently generated mice lacking Hic-5, which grew with no apparent abnormality (Kim-Kaneyama J, et al. J Mol Cell Cardiol. 2011;50(1):77-86). However, we discovered that recovery of arterial media following vascular injury is delayed significantly in Hic-5 knockout mice consequent to enhanced apoptosis of cultured vascular smooth muscle cells after mechanical stress; thus, Hic-5 is regarded as a novel factor in vascular remodeling. The Hic-5 gene is also induced by transforming growth factor-ß, a well-known accelerator in fibrosis. Hic-5 involvement in various fibrotic disorders, e.g., scar formation, keloid formation and glomerulosclerosis, has been proposed. siRNA silencing of Hic-5 in a breast cancer cell line reduces its invasiveness; moreover, Hic-5 serves as a steroid hormone co-activator and likely participates in endometriosis and prostate cancer. Thus, functional characterization of Hic-5 in various pathophysiological conditions may afford novel mechanistic insights into a wide variety of diseases.

m-Calpain induction in vascular endothelial cells on human and mouse atheromas and its roles in VE-cadherin disorganization and atherosclerosis.

BACKGROUND: Although dysfunction of VE-cadherin-mediated adherence junctions in vascular endothelial cells (ECs) is thought to be one of the initial steps of atherosclerosis, little is known regarding how VE-cadherin is disrupted during atherogenic development. This study focused on the role of calpain, an intracellular cysteine protease, in the proteolytic disorganization of VE-cadherin and subsequent progression of atherosclerosis. METHODS AND RESULTS: Increased expression of m-calpain was observed in aortic ECs in atherosclerotic lesions in humans and low-density lipoprotein receptor-deficient (ldlr(-/-)) mice. Furthermore, proteolytic disorganization of VE-cadherin was shown in aortic ECs in ldlr(-/-) and apolipoprotein E-deficient (apoE(-/-)) mice. Long-term administration of calpain inhibitors into these mice attenuated atherosclerotic lesion development and proinflammatory responses, as well as VE-cadherin disorganization, without normalization of plasma lipid profiles. Furthermore, in vivo transfection of m-calpain siRNA to ldlr(-/-) mice prevented disorganization of VE-cadherin and proatherogenic hyperpermeability in aortic ECs. Treatment of cultured ECs with oxidized LDL, lysophosphatidylcholine, or LDL pretreated with secreted phospholipase A(2) led to the induction of m-calpain but not of µ-calpain, thereby eliciting selective m-calpain overactivation. These data suggest that lysophosphatidylcholine-induced m-calpain directly cleaves a juxtamembrane region of VE-cadherin, resulting in dissociation of ß-catenin from the VE-cadherin complex, disorganization of adherence junctions, and hyperpermeability in ECs. CONCLUSIONS: Subtype-selective induction of m-calpain in aortic ECs during atherosclerotic progression is associated with proteolytic disorganization of VE-cadherin and proatherogenic hyperpermeability in cells. Thus, a strategy to selectively inhibit m-calpain may be useful for the therapeutic treatment of patients with atherosclerosis.

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.