Hidekazu Ota

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.

Molecular differences between sporadic serrated and conventional colorectal adenomas.

Purpose: The purpose is to compare the molecular characteristics of serrated adenomas (SAs) with those of conventional adenomas (CADs) and hyperplastic polyps (HPs). Experimental Design: We evaluated the proliferative activity and molecular alterations in 47 SAs (25 pure-type and 22 mixed-type), 71 CADs, and 23 HPs. Results: The proliferative activity of SAs, as evaluated by Ki-67 expression, was intermediate between CADs and HPs. There was no significant difference in the incidence of KRAS or p53 mutations between the three histological groups. In the microsatellite instability (MSI) analysis, 21% of SAs (9 of 43) showed MSI at two or more loci (MSI-H); corresponding values were 5% of CADs (3 of 64) and 8% of HPs (1 of 13; SAs versus CADs, P = 0.0125). MSI-H was more likely to be found in pure-type SAs (36%; 8 of 22) than in mixed-type SAs (5%; 1 of 21; P = 0.0212). Loss of hMLH-1 expression was found in 8 of 9 SAs with MSI-H. The incidence of BRAF or KRAS mutations was 36 and 15% of SAs, respectively; the combined incidence of BRAF and KRAS mutations occurred in 49% of SAs. However, there was no significant difference in the incidence of BRAF or KRAS mutations between SAs with and without MSI-H. Conclusions: Genetic instability is more frequently implicated in the tumorigenesis of SAs, especially pure-type SAs, than in that of CADs. In contrast, activation of the Ras/Raf/MEK/MAP kinase cascade by BRAF or KRAS mutation, independently of the genetic instability, may be associated with the progression of about half of SAs.

Cocaine-induced liver injury in mice is mediated by nitric oxide and reactive oxygen species

The modulating effects of nitric oxide (NO) and reactive oxygen species on cocaine-induced hepatotoxicity were examined by measuring plasma alanine aminotransferase activity and by carrying out histological studies. Liver injury was induced by a single injection of cocaine in adult male ICR mice. Pretreatment with aminoguanidine (an inhibitor of NO synthase), N-methyl-D-glucamine dithiocarbamate complex with iron ion (II) (Fe2+(MGD)2, a trapping reagent of NO) or deferoxamine complex with iron ion (III) (Fe3+-deferoxamine, a scavenger of NO) produced a marked inhibition of the hepatotoxicity induced by cocaine. In addition, pretreatment with allopurinol (an inhibitor of xanthine oxidase) and 1,3-dimethylthiourea (a scavenger of hydroxyl radical) also produced a potent inhibition. These findings suggest that a hydroxyl radical produced by the reaction of NO and superoxide anion (O2-) via peroxynitrite may be involved in the pathogenesis of cocaine hepatotoxicity.

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 A2 led to the induction of m-calpain but not of &mgr;-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 &bgr;-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.

Expression of Interleukin-17F in a Mouse Model of Allergic Asthma

Background: Interleukin (IL)-17F is a recently discovered cytokine and is derived from a panel of limited cell types, such as activated CD4+ T cells, basophils, and mast cells. IL-17F is known to induce several cytokines and chemokines. However, its involvement in airway inflammation has not been well understood. To this end, the expression of IL-17F and the inhibitory effects of glucocorticoids on its expression in a mouse model of asthma were examined. Methods: Five-week-old BALB/c male mice were sensitized by intraperitoneal injection (i.p.) of ovalbumin (OVA) with alum, and challenged by daily inhalation of aerosolized 1% OVA. 24 h after last challenge (OVA/OVA), the expression of IL-17F was examined in lung tissues by immunohistochemistry and reverse-transcription polymerase chain reaction. Control mice were sensitized and challenged with saline (Sham/Sham). In addition, a group of OVA-sensitized mice received i.p. injection of water-soluble dexamethasone (DEX) in saline 1 h before OVA challenge (OVA/DEX). Results: In sham-challenged mice, IL-17F was not expressed in the lungs, while, in contrast, IL-17F was predominantly expressed in bronchial epithelial cells in addition to the infiltrating inflammatory cells in OVA/OVA mice. Further, the expression of IL-17 F was significantly attenuated by the treatment of mice with DEX. Conclusion: These results suggest that bronchial epithelium-derived IL-17F may represent a new pharmacological target for glucocorticoids and may play a role in allergic asthma.

Preventive Effects of Heregulin-β1 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-&bgr;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-&bgr;1 levels were further decreased in accordance with the severity of coronary artery lesions. Expression of heregulin-&bgr;1 was observed at trace levels in intracoronary atherothrombosis obtained by aspiration thrombectomy from acute coronary syndrome patients. Heregulin-&bgr;1, but not heregulin-&agr;, 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-&bgr;1 significantly decreased endocytic uptake of [125I]acetylated low-density lipoprotein and ACAT activity, and increased cholesterol efflux to apolipoprotein (Apo)A-I from human macrophages. Chronic infusion of heregulin-&bgr;1 into ApoE−/− mice significantly suppressed the development of atherosclerotic lesions. Conclusions: This study provided the first evidence that heregulin-&bgr;1 inhibits atherogenesis and suppresses macrophage foam cell formation via SR-A and ACAT1 downregulation and ABCA1 upregulation.

Disappearance of CD21-positive Follicular Dendritic Cells Preceding the Transformation of Follicular Lymphoma: Immunohistological Study of the Transformation Using CD21, p53, Ki-67, and P-glycoprotein

Some follicular lymphomas histologically transform into diffuse aggressive lymphomas, the prognosis of which is poor. There are, however, no reliable histological criteria for predicting which cases will later undergo such transformation. In low-grade B-cell lymphomas, follicular dendritic cells form dense mesh-like networks that contain accumulating neoplastic B-cells. These are rare in high-grade lymphomas. We immunohistochemically analyzed CD21-positive follicular dendritic cells in 32 follicular lymphomas, including 3 transformed lymphomas, in addition to immunohistological study using P-glycoprotein, p53, and Ki-67. We found that the mesh-like networks in follicles are more clearly defined in low-grade lymphomas than in high-grade lymphomas (p = 0.015). Neoplastic follicles in 2 transformed lymphomas lost the networks of follicular dendritic cells before transformation despite the existence of morphologically clear follicles. This differed from the non-transformed cases of the same cytological grades. Prognosis was statistically better for patients with low-grade tumor than for those with high-grade tumor (p = 0.026), and there was a trend toward poorer survival among CD21-negative cases (p = 0.186). P-glycoprotein, p53, and Ki-67 expressions did not provide sufficient information to predict the transformation of follicular lymphoma. The presence of CD21-positive follicular dendritic cells in neoplastic follicles might help predict the potential of follicular lymphoma to transform to diffuse large B-cell lymphoma.

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-&bgr;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-&bgr;1 levels were further decreased in accordance with the severity of coronary artery lesions. Expression of heregulin-&bgr;1 was observed at trace levels in intracoronary atherothrombosis obtained by aspiration thrombectomy from acute coronary syndrome patients. Heregulin-&bgr;1, but not heregulin-&agr;, 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-&bgr;1 significantly decreased endocytic uptake of [125I]acetylated low-density lipoprotein and ACAT activity, and increased cholesterol efflux to apolipoprotein (Apo)A-I from human macrophages. Chronic infusion of heregulin-&bgr;1 into ApoE−/− mice significantly suppressed the development of atherosclerotic lesions. Conclusions: This study provided the first evidence that heregulin-&bgr;1 inhibits atherogenesis and suppresses macrophage foam cell formation via SR-A and ACAT1 downregulation and ABCA1 upregulation.

Transition from low-grade endometrial stromal sarcoma to high-grade endometrial stromal sarcoma.

We report on a case of a primary low-grade endometrial stromal sarcoma (ESS) that progressed to a secondary high-grade ESS. In the secondary tumor, the immunohistochemical profile and focal tumor cell proliferation pattern suggested that this tumor was not truly undifferentiated, but possessed features of endometrial stroma. Low-grade ESS of our patients primary tumor showed p53 protein overexpression, which is unusual in low-grade ESS, and her secondary high-grade ESS showed more prominent p53 immunoreactivity. This indicates that low-grade ESS that shows p53 immunoreactivity might progress to high-grade ESS, and it is considered that such cases of low-grade ESS should pay attention to the prognosis. Immunoreactivity for epidermal growth factor receptor was observed in both tumors, suggesting a relationship between the primary and secondary tumors in our case. Further study requires more immunohistochemical data for cases in which low-grade ESS transitions to high-grade ESS; in particular, data on epidermal growth factor receptor expression are necessary to define new therapeutic strategies for ESS.

Expression of CD10 and cytokeratins in ovarian and renal clear cell carcinoma.

A solid pattern of tumor cells with a clear cytoplasm is common to both ovarian clear cell carcinoma (OvCCC) and renal clear cell carcinoma (RCCC). This study examined the possible differential expression of CD10 and cytokeratins (CK7, CK20, 34βE12, and CAM5.2) between these two types. An immunohistochemical technique using peroxidase-labeled amino acid polymers was used to test formalin-fixed and paraffin-embedded tissues. In OvCCC, 6 of 29 cases were positive for CD10, and all cases had expression of CK7, 34βE12, and CAM5.2. In contrast, all 24 RCCC cases had CD10 and CAM5.2 immunoreactivity, but none had any staining for 34βE12. CK7 was only expressed in nine cases. No CK20 positivity was observed in any sample from either tumor type. Localization of CD10 expression was different in OvCCC versus RCCC. Although positive staining for 34βE12 strongly suggests OvCCC, sometimes only a few cells may be stained. Therefore, 34βE12-negative biopsies also should be evaluated for CD10 and CK7 immunoreactivity to enable histologic and cytologic differential diagnosis.