15-Lipoxygenase-2 is expressed in macrophages in human carotid plaques and regulated by hypoxia-inducible factor-1alpha.

BACKGROUND: Macrophages are prominent in hypoxic areas of atherosclerotic lesions and their secreted cytokines, growth factors and activity of enzymes are involved in atherogenesis. Previously, we showed that 15-lipoxygenase (LOX)-2 is expressed in human monocyte-derived macrophages and that hypoxia increases 15-LOX-2 expression and secretion of pro-inflammatory molecules. Here we investigated whether human carotid plaque macrophages express 15-LOX-2 and whether its expression in macrophages is regulated by hypoxia through hypoxia-inducible factor 1alpha (HIF-1alpha). MATERIALS AND METHODS: Carotid plaques from 47 patients with high-grade symptomatic carotid artery stenosis were analysed using immunohistochemistry, and stained areas were quantified by digital image analysis. Carotid plaque macrophages were isolated with anti-CD14 immunobeads using an immunomagnetic bead technique. Primary macrophages were transfected with HIF-1alpha siRNA or control siRNA before extraction of RNA and medium analysis. RESULTS: In paired tissue sections, the extent of staining for CD68 correlated with staining for 15-LOX-2 but not for 15-LOX-1. In carotid plaque macrophages isolated with anti-CD14 immunobeads, 15-LOX-2 mRNA was expressed at high levels. In primary macrophages, 15-LOX-2 expression was significantly increased by incubation with the HIF-1alpha stabilizer dimethyloxalylglycine. Knockdown of HIF-1alpha significantly decreased production of the 15-LOX-2 enzyme products 12- and 15-hydroxyeicosatetraenoic acid. In carotid plaques, HIF-1alpha staining correlated with staining for 15-LOX-2. CONCLUSIONS: These results demonstrate that 15-LOX-2 is highly expressed in human plaques and is correlated with the presence of macrophages and HIF-1alpha. 15-LOX-2 enzyme activity can be modulated by HIF-1alpha. Thus, increased expression of 15-LOX-2 in macrophages in hypoxic atherosclerotic plaque may enhance inflammation and the recruitment of inflammatory cells.

Circulating matrix metalloproteinase 9 levels in relation to sampling methods, femoral and carotid atherosclerosis.

OBJECTIVES: To examine whether circulating levels of matrix metalloproteinase 9 (MMP-9) were associated with ultrasound-assessed intima-media thickness (IMT) and echolucent plaques in the carotid and femoral arteries. To examine preanalytical sources of variability in MMP-9 concentrations related to sampling procedures. SUBJECTS AND DESIGN: Plasma and serum MMP-9 levels were compared with ultrasound assessed measures of femoral and carotid atherosclerosis, in a cross-sectional study of 61-year-old men (n = 473). Preanalytical sources of variability in MMP-9 levels were examined in 10 healthy subjects. Main outcome measures were circulating levels of MMP-9 in serum and plasma, IMT of the carotid and femoral arteries, and plaque status based on size and echolucency. SETTING: Research unit at university hospital. RESULTS: Plasma concentrations of total and active MMP-9 were associated with femoral artery IMT independently of traditional cardiovascular risk factors, and were higher in subjects with moderate to large femoral plaques. Plasma MMP-9 concentration was higher in men with echolucent femoral plaques (P = 0.006) compared with subjects without femoral plaques. No similar associations were found for carotid plaques. MMP-9 concentrations were higher in serum than in plasma, and higher when sampling was performed with Vacutainer than with syringe. MMP-9 levels in serum were more strongly associated with peripheral neutrophil count compared with MMP-9 levels in plasma. CONCLUSIONS: Plasma MMP-9 levels were associated with atherosclerosis in the femoral artery, and total MMP-9 concentration was higher in men with echolucent femoral plaques. The choice of sample material and sampling method affect the measurements of circulating MMP-9 levels.

Identification of transient glycosylation alterations of sialylated mucin oligosaccharides during infection by the rat intestinal parasite Nippostrongylus brasiliensis.

The sialylation of the oligosaccharides from small-intestinal mucins during a 13-day infectious cycle was studied in Sprague-Dawley rats with the parasite Nippostrongylus brasiliensis. Sialic acid analysis and release, permethylation and analysis by GC-MS of the sialylated oligosaccharides isolated from the 'insoluble' mucin complex revealed a relative decrease (4-7-fold) of N-glycolylneuraminic acid compared with N-acetylneuraminic acid just before parasite expulsion. Northern blots showed that this effect was due to the decreased expression of a hydroxylase converting CMP-N-acetylneuraminic acid into CMP-N-glycolylneuraminic acid. Analysis of other rat strains showed that this parasite infection also caused the same effect in these animals. Detailed analysis of infected Sprague-Dawley rats revealed four sialylated oligosaccharides not found in the uninfected animals. These new oligosaccharides were characterized in detail and all shown to contain the trisaccharide epitope NeuAc/NeuGcalpha2-3(GalNAcbeta1-4)Galbeta1 (where NeuGc is N-glycolyl neuraminic acid). This epitope is similar to the Sd(a)- and Cad-type blood-group antigens and suggests that the infection causes the induction of a GalNAcbeta1-4 glycosyltransferase. This model for an intestinal infection suggests that the glycosylation of intestinal mucins is a dynamic process being modulated by the expression of specific enzymes during an infection process.

Excretion of hepatitis A virus (HAV) in adults: comparison of immunologic and molecular detection methods and relationship between HAV positivity and infectivity in tamarins.

Fecal excretion of hepatitis A virus (HAV) in 18 patients with HAV infection was evaluated by enzyme immunoassay (EIA) to detect viral antigen and by reverse transcription-PCR amplification followed by ethidium bromide staining (PCR-ETBr) or nucleic acid hybridization (PCR-NA) to detect viral genetic material. A gradation of sensitivity was observed in the detection of virus by the three methods. In persons who had detectable virus, serial stool samples were found to be positive by EIA for up to 24 days after the peak elevation of liver enzymes. Viral genetic material could be detected by PCR-ETBr for up to 34 days and by PCR-NA for up to 54 days after the peak elevation of liver enzymes. After intravenous inoculation of tamarins with stool suspensions categorized as highly reactive for HAV (positive by EIA, PCR-ETBr, and PCR-NA), moderately reactive (positive by PCR-ETBr and PCR-NA), or weakly reactive (positive by PCR-NA), only tamarins infected with highly reactive stool suspensions (EIA positive) developed HAV infection. We conclude that positivity of stool specimens for HAV by PCR-ETBr or PCR-NA indicates a lower potential for infectivity, compared to that of EIA-positive stools.

Significance of symptoms and signs in patients with traumatic aortic rupture.

The records of 50 patients with traumatic aortic rupture (Group I) and 50 patients with blunt chest trauma but negative aortograms (Group II) were reviewed retrospectively. Symptoms and signs referable to the chest and thoracic aorta were recorded and compared in Group I and Group II patients. Each patient's chart was evaluated for chest pain, respiratory distress, thoracic back pain, hypotension, hypertension, and decreased femoral pulses. None of the symptoms or signs attained statistical significance between Group I and Group II patients. The only significant difference between Group I and Group II patients was in the injury severity score (ISS). The mean ISS for aortic rupture patients was 42.1 +/- 11.6 (SD), but was only 19.9 +/- 11.4 (SD) (P less than .001) for patients without aortic rupture. We conclude that the diagnosis of aortic rupture in patients sustaining blunt chest trauma cannot be accurately predicted or excluded on the basis of the patients' presenting complaints or physical findings.

The management of subclavian artery injuries following blunt thoracic trauma.

The records of 15 patients who sustained blunt rupture of the subclavian artery were reviewed. The findings on physical examination included arterial hypotension, unilateral absence of the radial pulse, brachial plexus palsy, and supraclavicular hematoma. The chest roentgenographic findings included wide mediastinums, apical pleural hematomas, and first rib fractures. Fourteen patients survived to undergo angiography and operation. Arterial continuity was restored by primary anastomosis, synthetic grafts, and venous interposition grafts. Ligation of a pseudoaneurysm was carried out in 1 patient with a complete brachial plexus palsy. Amputation of an upper extremity was required in 1 patient. Two patients died postoperatively. We conclude that blunt subclavian artery injuries may be suspected clinically. Absent upper extremity pulses, a wide mediastinum, unrelenting thoracic hemorrhage, and persistent hypotension dictate the necessity for aortography. Relative indications for angiography include brachial plexus palsy, apical pleural hematoma, and a fractured first rib.

Chest roentgenographic findings in 26 patients with traumatic rupture of the thoracic aorta.

The initial 100-cm anteroposterior (AP) supine chest roentgenograms of 26 patients with proven traumatic aortic rupture were reviewed. The 20 male patients and six female patients ranged in age from 12 to 75 years. Distortion of normal aortic contour or blurring of the aortic outline occurred in 23 cases. Opacification of the radiolucent space between the aorta and pulmonary artery was seen in 22 instances. The mean mediastinal width, measured at the superior border of the anterior fourth rib, was 9.4 cm. Obliteration of the medial aspect of the left upper lung field was observed on 13 films. Obliteration of the shadow of the descending aorta, hemopneumothorax, and tracheal shift to the right each occurred in 17, 14, and 12 cases, respectively. This study illustrates that distortion or blurring of the aortic arch contour, opacification of the clear space between the aorta and pulmonary artery, and increased mediastinal width (mean = 9.4 cm) are the most frequently occurring abnormalities on the initial AP supine chest films of patients with traumatic aortic rupture. Such findings should arouse the suspicion of aortic rupture, and warrant aortography.

Characterization, toxicity and therapeutic efficacy of adriamycin encapsulated in liposomes.

The inherent toxicities of drug-free liposomes were compared. Positively charged liposomes were more toxic in vitro (chick heart cells) and in vivo (5-fold reduction in LD50 in mice) than neutral or negatively charged liposomes. Based on these findings, adriamycin was encapsulated in negatively charged liposomes (PG: PC: Chol--1:4:5), sequentially extruded through polycarbonate membranes (to 0.2 micrometer pore size) and purified by exhaustive dialysis. This preparation had a mean vesicle diameter of 0.24 micrometer and was stable in serum (24 hr drug retention of 85%). As compared with free drug, liposome-encapsulated adriamycin was less toxic in vitro to chick heart cells. In mice, adriamycin displayed short- (4-14 day) and long- (45-70 day) term toxicity. Encapsulating adriamycin increased both short- (20-50 mg/kg) and long- (10-15 mg/kg to 25-30 mg/kg) term LD50 levels. As compared with free drug, administering encapsulated adriamycin in vivo reduced the incidence of cardiac histopathologic lesions at 20 and 40 mg/kg. Compared in vivo, plasma levels of liposome-encapsulated adriamycin were 2/3-fold higher than free drug up to 24 hr post drug administration. Cardiac uptake of adriamycin was reduced 2-fold (conc x time value for 48 h) following encapsulated drug administration. Encapsulating adriamycin in liposomes did not alter its therapeutic effect against L1210 leukemic cells in vivo.

Developmental pattern of cAMP, adenyl cyclase, and cAMP phosphodiesterase in the palate, lung, and liver of the fetal mouse: alterations resulting from exposure to methylmercury at levels inhibiting palate closure.

Exposure to methylmercury (MeHg: 10 mg Hg/kg maternal body weight) on 12(6) (days hours) of gestation significantly delays palate closure in the Swiss Webster CFW mouse. The cAMP content and activity of adenyl cyclase and phosphodiesterase (PDE) were measured in the tissues of control and MeHg-induced cleft palates between 13(6) and 17(6) of gestation. Lung and liver were investigated similarly to determine if MeHg affected the adenyl cyclase system of the palate in a unique manner. In control palatal tissue, cAMP levels increased sharply from 13(22) (undetectable) to 14(6) (maximum). PDE activity increased similarly up to 14(2), but decreased 50% between 14(2) and 14(6). Since it has been reported that cAMP induces the synthesis of PDE, the difference in cAMP/PDE from 13(22) to 14(2) and from 14(2) to 14(6) suggests the localization of relatively high levels of cAMP in at least two separate compartments. Between 14(6) and 14(10), the adenyl cyclase activity of control palates decreased significantly. This rapid decrease suggests relatively high adenyl cyclase activity in the medial edge epithelial cells which undergo autolysis prior to shelf fusion (centered at 14(15). Maternal MeHg administration at 12(6) delayed the median time of palatal shelf rotation (14(13)) by 5 hours, and significantly altered the developmental pattern of the adenyl cyclase system. Thus, the increase in cAMP between 14(2) and 14(6) was abolished and the decrease in adenyl cyclase activity between 14(6) and 14(10) was delayed by almost 20 hours. These changes may be manifestions of a MeHg-induced delay in medial edge epithelial cell differentiation. In a previous study, we observed that the fetal liver exhibits the highest MeHg concentration of all tissues. Since MeHg only slightly altered the adenyl cyclase system of the fetal liver compared to the lung and palate (in which MeHg uptake is considerably less), it may be that the effects of MeHg on palatal tissue are not due to a direct effect of MeHg on components of the adenyl cyclase system.

Preparation of unilamellar liposomes of intermediate size (0.1-0.2 mumol) by a combination of reverse phase evaporation and extrusion through polycarbonate membranes.

Liposomes can be prepared by a combination of reverse phase evaporation and sequential extrusion through polycarbonate membranes. The vesicles have diameters in the range 0.05-0.5 micron and are mostly unilamellar as indicated by electon microscopy, capture volume, and availability of reactive groups to periodate oxidation. Sequential extrusion leads to a decrease in the encapsulation efficiency by 2-4-fold, depending upon the lipid composition. The inclusion of cholesterol at a 1 : 1 molar ratio of cholesterol-to-phospholipid increases both the mean size and the size heterogeneity of the liposomes as measured by negative-stain electron microscopy. The mean size of vesicles with an equal molar ratio of cholesterol-to-phospholipid after extrusion through a 0.1 micron membrane is 0.140 micron. Vesicles composed of phosphatidylglycerol/phosphatidylcholine (1 : 4) have a mean size of 0.08 micron after extrusion through a 0.1 micron membrane. The intermediate-size (0.1-0.2 micron) vesicles formed by this process have an aqueous space-to-lipid ratio of 3 : 5 and capture between 12 and 25% of the aqueous phase. The procedure is relatively simple, rapid, and yields almost quantitative recovery of vesicles that encapsulate a large percentage of the total aqueous space.

Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes.

Liposomes of defined size and homogeneity have been prepared by sequential extrusion of the usual multilamellar vesicles through polycarbonate membranes. The process is easy, reproducible, produces no detectable degradation of the phospholipids, and can double the encapsulation efficiency of the liposome preparation. Multilamellar vesicles extruded by this technique are shown by both negative stain and freeze-fracture electron microscopy to have mean diameters approaching the pore diameter of the polycarbonate membrane through which they were extruded. When sequentially extruded down through a 0.2 micron membrane, the resulting vesicles exhibit a very homogeneous size distribution with a mean diameter of 0.27 micron while maintaining an acceptable level of encapsulation of the aqueous phase.

Effects of methyl mercury on murine fetal amino acid uptake, protein synthesis and palate closure.

Methylmercury (MeHg: 5 mg Hg/kg maternal body weight) in 0.13 M NaCl, 0.01 M NaH2PO4-Na2HPO4, pH 7.4 (PBS) administered to gravid CFW mice on day 12, hour 6 (12(6)) of gestation induced a high incidence of cleft palate in fetuses examined on days 15(6) (72%), 16(6) (62%) and 17(6) (40%). Palate closure (100%) in PBS control animals occurred by 14(10). One day post MeHg administration, total fetal protein was decreased 22% while DNA content was unaltered. Protein was maximally decreased (28%) on 14(6) and, thereafter, returned toward control levels. Alterations in DNA content followed a similar pattern; but the maximal decrease (32%) occurred on 15(6). The rate of fetal protein synthesis was depressed 5% at 12(9) and between 20% to 26% from this time to 13(6) (end of observation). The agreement between the calculated decrease in protein synthesis (19%) and the measured decrease in protein content (22%) suggests that a reduction in protein synthesis is responsible for the decreased fetal protein content. Placental blood flow and fetal water space, measured with 3H--H2O at 12(18), were not affected by MeHg treatment. However, fetal free amino acid concentrations at 12(18) were generally decreased (alanine, 23.0%; valine, 9.7%; methionine, 22.6%; isoleucine, 12.0%; leucine, 18.2%) while uptake of the non-metabolizable amino acid, 14C-cycloleucine, was decreased 23%. From this, it is concluded that the growth inhibitory effects of MeHg are related, at least in part, to impaired placental/fetal transfer of amino acids.