V. Jukka O. Laine

Angiopoietin-regulated recruitment of vascular smooth muscle cells by endothelial-derived heparin binding EGF-like growth factor

Recruitment of vascular smooth muscle cells (SMC) by endothelial cells (EC) is essential for angiogenesis. Endothelial‐derived heparin binding EGF‐like growth factor (HB‐EGF) was shown to mediate this process by signaling via ErbB1 and ErbB2 receptors in SMCs. 1) Analysis of ErbB‐ligands demonstrated that primary ECs expressed only HB‐EGF and neuregulin‐1. 2) Primary SMCs expressed ErbB1 and ErbB2, but not ErbB3 or ErbB4. 3) Consistent with their known receptor specificities, recombinant HB‐EGF, but not neuregulin‐1, stimulated tyrosine phosphorylation of ErbB1 and ErbB2 and migration in SMCs. 4) Neutralization of HB‐EGF or inhibition of ErbB1 or ErbB2 blocked 70–90% of the potential of ECs to stimulate SMC migration. Moreover, 5) angiopoietin‐1, an EC effector with a role in recruitment of SMC‐like cells to vascular structures in vivo, enhanced EC‐stimulated SMC migration by a mechanism involving up‐regulation of endothelial HB‐EGF. Finally, 6) immunohistochemical analysis of developing human tissues demonstrated that HB‐EGF was expressed in vivo in ECs associated with SMCs or pericytes but not in ECs of the hyaloid vessels not associated with SMCs. These results suggest an important role for HB‐EGF and ErbB receptors in the recruitment of SMCs by ECs and elaborate on the mechanism by which angiopoietins exert their vascular effects.—Iivanainen E., Neli‐markka L., Elenius V., Heikkinen S.‐M., Junttila T. T., Sihombing L., Sundvall M., Määttä J. A., Laine V. J. O., Ylä‐Herttuala S., Higashiyama S., Alitalo K., Elenius K. Angiopoietin‐regulated recruitment of vascular smooth muscle cells by endothelial‐derived heparin binding EGF‐like growth factor. FASEB J. 17, 1609–1621 (2003)

Non-specific binding of [18F]FDG to calcifications in atherosclerotic plaques: experimental study of mouse and human arteries.

Purpose[18F]FDG has been used as an inflammation marker and shown to accumulate in inflammatory atherosclerotic plaques. The aim of this study was to investigate the uptake and location of [18F]FDG in atherosclerotic plaque compartments.MethodsThe biodistribution of intravenously administered [18F]FDG was analysed in atherosclerotic LDLR/ApoB48 mice (n=11) and control mice (n=9). Digital autoradiography was used to detect the ex vivo distribution in frozen aortic sections. In vitro binding of [18F]FDG in human atherosclerotic arteries was also examined.ResultsThe uptake of [18F]FDG was significantly higher in the aorta of atherosclerotic mice as compared with the control mice. Autoradiography of excised arteries showed higher [18F]FDG uptake in the plaques than in the healthy vessel wall (mean ratio ±SD 2.7±1.1). The uptake of [18F]FDG in the necrotic, calcified sites of the advanced atherosclerotic lesions was 6.2±3.2 times higher than that in the healthy vessel wall. The in vitro studies of human arterial sections showed marked binding of [18F]FDG to the calcifications but not to other structures of the artery wall.ConclusionIn agreement with previous studies, we observed [18F]FDG uptake in atherosclerotic plaques. However, prominent non-specific binding to calcified structures was found. This finding warrants further studies to clarify the significance of this non-specific binding in human plaques in vivo.

Uptake of inflammatory cell marker [11C]PK11195 into mouse atherosclerotic plaques.

PurposeThe ligand [11C]PK11195 binds with high affinity and selectivity to peripheral benzodiazepine receptor, expressed in high amounts in macrophages. In humans, [11C]PK11195 has been used successfully for the in vivo imaging of inflammatory processes of brain tissue. The purpose of this study was to explore the feasibility of [11C]PK11195 in imaging inflammation in the atherosclerotic plaques.MethodsThe presence of PK11195 binding sites in the atherosclerotic plaques was verified by examining the in vitro binding of [3H]PK11195 onto mouse aortic sections. Uptake of intravenously administered [11C]PK11195 was studied ex vivo in excised tissue samples and aortic sections of a LDLR/ApoB48 atherosclerotic mice. Accumulation of the tracer was compared between the atherosclerotic plaques and non-atherosclerotic arterial sites by autoradiography and histological analyses.ResultsThe [3H]PK11195 was found to bind to both the atherosclerotic plaques and the healthy wall. The autoradiography analysis revealed that the uptake of [11C]PK11195 to inflamed regions in plaques was more prominent (p = 0.011) than to non-inflamed plaque regions, but overall it was not higher than the uptake to the healthy vessel wall. Also, the accumulation of 11C radioactivity into the aorta of the atherosclerotic mice was not increased compared to the healthy control mice.ConclusionsOur results indicate that the uptake of [11C]PK11195 is higher in inflamed atherosclerotic plaques containing a large number of inflammatory cells than in the non-inflamed plaques. However, the tracer uptake to other structures of the artery wall was also prominent and may limit the use of [11C]PK11195 in clinical imaging of atherosclerotic plaques.

Effects of Age, Diet, and Type 2 Diabetes on the Development and FDG Uptake of Atherosclerotic Plaques

OBJECTIVES This study investigated the effects of age, duration of a high-fat diet, and type 2 diabetes on atherosclerotic plaque development and uptake of (18)F-fluorodeoxyglucose ((18)F-FDG) in 2 mouse models. BACKGROUND The animals age and start time and duration of a high-fat diet have effects on plaque composition in atherosclerotic mice. METHODS The aortas of atherosclerotic low-density lipoprotein receptor deficient mice expressing only apolipoprotein B100 (LDLR(-/-)ApoB(100/100)) and atherosclerotic and diabetic mice overexpressing insulin-like growth factor II (IGF-II/LDLR(-/-)ApoB(100/100)) were investigated at 4, 6, and 12 months of age and older after varying durations of high-fat diet. C57BL/6N mice on normal chow served as controls. Plaque size (intima-to-media ratio), macrophage density (Mac-3 staining), and plaque uptake of (18)F-FDG were studied by means of in vivo positron emission tomography/computed tomography by ex vivo autoradiography and by histological and immunohistochemical methods. RESULTS From the ages of 4 to 6 months and 12 months and older, the plaque size increased and the macrophage density decreased. Compared with the controls, the in vivo imaging showed increased aortic (18)F-FDG uptake at 4 and 6 months, but not at 12 months and older. Autoradiography showed focal (18)F-FDG uptake in plaques at all time points (average plaque-to-normal vessel wall ratio: 2.4 ± 0.4, p < 0.001) with the highest uptake in plaques with high macrophage density. There were no differences in the plaque size, macrophage density, or uptake of (18)F-FDG between LDLR(-/-)ApoB(100/100) and IGF-II/LDLR(-/-)ApoB(100/100) mice at any time point. CONCLUSIONS The 6-month-old LDLR(-/-)ApoB(100/100) and IGF-II/LDLR(-/-)ApoB(100/100) mice demonstrated highly inflamed, large, and extensive atherosclerotic plaques after 4 months of a high-fat diet, presenting a suitable model for studying the imaging of atherosclerotic plaque inflammation with (18)F-FDG. The presence of type 2 diabetes did not confound evaluation of plaque inflammation with (18)F-FDG.

Detection of Hypoxia by [18F]EF5 in Atherosclerotic Plaques in Mice

Objective—Atherosclerotic plaques with large lipid cores and inflammation contain regions of hypoxia. We examined the uptake of 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide ([18F]EF5), a specific marker of hypoxia labeled for positron emission tomography, in mouse atherosclerotic plaques. Methods and Results—Atherosclerotic mice of 2 different genetic backgrounds (low-density lipoprotein receptor−/− apolipoprotein B100/100 and insulin-like growth factor II/low-density lipoprotein receptor−/− apolipoprotein B100/100) were first fed a Western diet to induce development of plaques with variable phenotypes and then injected with [18F]EF5. C57BL/6N mice served as controls. Aortas were dissected for biodistribution studies, autoradiography, histology, and immunohistochemistry. Uptake of [18F]EF5 was significantly higher in the aortas of mice with large atherosclerotic plaques than in the C57BL/6N controls. Furthermore, autoradiography demonstrated, on average, 2.0-fold higher [18F]EF5 uptake in atherosclerotic plaques than in the adjacent normal vessel wall. Hypoxia in plaques was verified by using an EF5 adduct-specific antibody and pimonidazole. The blood clearance of [18F]EF5 was slow, with blood radioactivity remaining relatively high up to 180 minutes after injection. Conclusion—Large atherosclerotic plaques in mice contained hypoxic areas and showed uptake of [18F]EF5. Despite its slow blood clearance, the high uptake of [18F]EF5 in plaques suggested that plaque hypoxia is a potential target for identifying high-risk plaques noninvasively.

Antibacterial effects of human group IIA and group XIIA phospholipase A2 against Helicobacter pylori in vitro

Group IIA phospholipase A2 (PLA2‐IIA) is an enzyme which has important roles in inflammation and infection. Recently, a novel human secretory PLA2 called group XIIA PLA2 (PLA2‐XIIA) has been identified. Both PLA2‐IIA and PLA2‐XIIA are bactericidal against Gram‐positive bacteria like many other secretory PLA2s. However, PLA2‐XIIA is the only known PLA2 displaying significant bactericidal activity against the Gram‐negative bacterium Escherichia coli. We examined the antibacterial properties of recombinant human PLA2‐IIA and PLA2‐XIIA against Helicobacter pylori, a Gram‐negative bacterium, in vitro. PLA2‐IIA was not bactericidal against H. pylori, whereas PLA2‐XIIA effectively killed H. pylori at a concentration of 50 μg/ml but was not bactericidal at concentrations of 0.5 μg/ml and 5 μg/ml.

Single doses of FK506 and OKT3 reduce severity in early experimental acute pancreatitis

OBJECTIVE To find out if two immunomodulatory drugs used in organ transplantation (FK506 (tacrolimus) and OKT3 (Orthoclone) would reduce early inflammatory complications in experimental acute pancreatitis. DESIGN Laboratory study. SETTING University hospital, Germany. ANIMALS 36 Balb/c mice. INTERVENTIONS Pancreatitis induced by 7 intraperitoneal injections of cerulein 50 microg/kg at hourly intervals followed by FK506 0.32 mg/kg, OKT3 0.6 mg/kg, or 0.9% sodium chloride (controls) (n = 12 in each group). 12 hours after induction of pancreatitis the animals were killed. MAIN OUTCOME MEASURES Serum amylase activity and interleukin-6 (IL-6) concentrations; histological damage to pancreas and lungs, apoptotic cells in pancreas; and myeloperoxidase activity in lungs. RESULTS No animal died during the experiment. At 12h serum amylase activity and IL-6 concentrations were increased in all 3 groups, but highest in the OKT3 group. The pancreatic histological score, apoptosis, and inflammatory infiltration were lower in the two experimental groups than controls, but the degree of vacuolisation of acinar cells was similar. Packed cell volume was higher in the control than the experimental groups, and pulmonary damage and myeloperoxidase activity were less in the experimental groups than the controls. CONCLUSION Single therapeutic doses of FK506 and OKT3 reduced the early severity of pancreatitis, pulmonary damage, and haemoconcentration in mice. Single doses of FK506 or OKT3 may therefore be effective in preventing the early complications of pancreatitis.

Uptake of 68gallium in atherosclerotic plaques in LDLR-/-ApoB100/100 mice

BackgroundAtherosclerosis is a chronic inflammatory disease of artery wall characterized by infiltration of monocytes into subendothelial space and their differentiation into macrophages. Since rupture-prone plaques commonly contain high amounts of activated macrophages, imaging of the macrophage content may provide a useful tool for the evaluation of plaque vulnerability. The purpose of this study was to explore the uptake of 68gallium (68Ga) in atherosclerotic plaques in mice.MethodsUptake of ionic 68Ga was investigated in atherosclerotic LDLR-/-ApoB100/100 and C57BL/6N control mice at 3 h after injection. The ex vivo biodistribution of the 68Ga was assessed and autoradiography of aortic cryosections was defined. In vivo imaging of 68Ga was performed using a small animal positron emission tomography PET/CT scanner.ResultsOur results revealed that the uptake of 68Ga-radioactivity was higher in atherosclerotic plaques than in healthy vessel wall (ratio 1.8 ± 0.2, p = 0.0002) and adventitia (ratio 1.3 ± 0.2, p = 0.0011). The autoradiography signal co-localized with macrophages prominently as demonstrated by Mac-3 staining. In both mice strains, the highest level of radioactivity was found in the blood.ConclusionsWe observed a moderate but significantly elevated 68Ga-radioactivity uptake in the aortic plaques of atherosclerotic mice, especially at the sites rich in macrophages. While the uptake of 68Ga was promising in this animal model, the slow blood clearance may limit the usability of 68Ga as a PET tracer for clinical imaging of atherosclerotic plaques.

Expression of Human Group II Phospholipase A2 in Transgenic Mice

Group II phospholipase A2 (PLA2) has been proposed to play an important role in inflammation and defense against bacterial infection. We investigated tissues of transgenic mice expressing the human group II PLA2 gene by immunohistochemistry using rabbit anti-human group II PLA2 antibodies, and by in situ hybridization by probing with human group II PLA2 mRNA anti-sense (test) and sense (control) riboprobes. By immunohis-tochemistry, human group II PLA2 was found in various mouse tissues and cell types including hepatocytes, proximal tubule cells of the kidney, epithelial cells of the renal pelvis, urinary bladder and ureter, granulosa cells of Graafian follicles, aortic intima and media, cartilage, epiphyseal bone, bronchial epithelial cells, and connective tissue cells in the dermis. By in situ hybridization, group II PLA2 mRNA was localized in hepatocytes, epidermal cells, dermal cells, connective tissue fibroblasts, epithelial and smooth muscle cells of the urinary bladder, and cells of Bowmans capsule. These results show that human group II PLA2 is expressed in large amounts in hepatocytes and many extrahepatic tissues of the transgenic mice. These animals provide a useful new tool for studies on the metabolism, in vivo effects, and physiological and pathological roles of phospholipase A2. (J Histochem Cytochem 45:1109–1119, 1997)

Maternal hyperglycemia leads to fetal cardiac hyperplasia and dysfunction in a rat model

Accelerated fetal myocardial growth with altered cardiac function is a well-documented complication of human diabetic pregnancy, but its pathophysiology is still largely unknown. Our aim was to explore the mechanisms of fetal cardiac remodeling and cardiovascular hemodynamics in a rat model of maternal pregestational streptozotocin-induced hyperglycemia. The hyperglycemic group comprised 107 fetuses (10 dams) and the control group 219 fetuses (20 dams). Fetal cardiac function was assessed serially by Doppler ultrasonography. Fetal cardiac to thoracic area ratio, newborn heart weight, myocardial cell proliferative and apoptotic activities, and cardiac gene expression patterns were determined. Maternal hyperglycemia was associated with increased cardiac size, proliferative, apoptotic and mitotic activities, upregulation of genes encoding A- and B-type natriuretic peptides, myosin heavy chain types 2 and 3, uncoupling proteins 2 and 3, and the angiogenetic tumor necrosis factor receptor superfamily member 12A. The genes encoding Kv channel-interacting protein 2, a regulator of electrical cardiac phenotype, and the insulin-regulated glucose transporter 4 were downregulated. The heart rate was lower in fetuses of hyperglycemic dams. At 13-14 gestational days, 98% of fetuses of hyperglycemic dams had holosystolic atrioventricular valve regurgitation and decreased outflow mean velocity, indicating diminished cardiac output. Maternal hyperglycemia may lead to accelerated fetal myocardial growth by cardiomyocyte hyperplasia. In fetuses of hyperglycemic dams, expression of key genes that control and regulate cardiomyocyte electrophysiological properties, contractility, and metabolism are altered and may lead to major functional and clinical implications on the fetal heart.