Elevated tissue expression of thrombomodulatory factors correlates with acute symptomatic carotid plaque phenotype.

OBJECTIVES: Thrombomodulatory factors have been implicated in plaque instability. The aim was to examine the relationship between thrombomodulatory gene expression, timing of clinical events and plaque histology. DESIGN OF STUDY: Plaques were obtained from 40 consecutive patients undergoing carotid endarterectomy and divided into three groups (group 1, early symptomatic, within 1 month; group 2, late symptomatic, 1-6 months and group 3, asymptomatic). Total RNA was isolated to determine the expression of tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), plasminogen activator inhibitor-1 (PAI-1), tissue factor (TF), tissue factor pathway inhibitor (TFPI), thrombomodulin (TM), CD68 and vascular endothelial-cadherin (VE-Cadherin). RESULTS: Expression of t-PA, PAI-1, TF, TFPI, TM, CD68 and VE-cadherin were significantly increased in the early symptomatic group (p=0.019, 0.028, 0.018, 0.025, 0.038, 0.016 and 0.027 respectively), but the level of gene expression in the late symptomatic group was indistinguishable from the asymptomatic group. The incidence of plaque rupture and intraplaque haemorrhage was significantly increased in the early symptomatic groups (58% versus 18%/18% group 2/3, and 55% versus 6%/9% respectively, p<0.05 for both). CONCLUSIONS: Expression of thrombomodulatory genes is increased in unstable plaques, though levels after 1 month are comparable to asymptomatic plaques. This transient rise may influence plaque instability, and rapid resolution mirrors the clinical reduction in risk of further thrombo-embolic events.

The placental bed in pregnancies complicated by primary antiphospholipid syndrome.

UNLABELLED: Pregnancy in women with primary antiphospholipid syndrome (APS) is frequently associated with placental insufficiency leading to intrauterine growth restriction (IUGR)+/-fetal death, pre-eclampsia, placental abruption, premature delivery or thrombosis. The aim of this study was to investigate the placental bed in APS pregnancies for evidence of impaired trophoblast invasion, endothelial cell activation (ECA) and macrophage infiltration. METHODS: Biopsies from the presumed site of the placental bed were obtained from 12 women with treated primary APS and 16 controls. Immunohistochemical methods were employed to investigate expression of cytokeratin (trophoblasts), alpha-actin (smooth muscle), CD68 (macrophages) and VCAM-1 (as marker of ECA). Fibrinoid and elastin distribution and expression were determined by periodic acid/Schiff and orcein stain, respectively. RESULTS: Three APS pregnancies developed IUGR, one with concurrent pre-eclampsia. Eight of 12 APS biopsies were confirmed to be from the placental bed; one patient failed to meet APS criteria and was excluded from analysis; six included spiral arteries in the biopsy; 11 of 16 controls' biopsies were from the placental bed. APS biopsies had a higher concentration of inflammatory cells (p=0.0001), particularly macrophages (p=0.014). Three APS biopsies showed necrosis with hyperplastic vessels; one demonstrated arterial thromboses, but none had profound vasculopathy/atherosis or ECA. CONCLUSION: Inflammatory mechanisms in the placental bed may contribute to APS pregnancy complications.

Enhanced vascular responses to adrenomedullin in mice overexpressing receptor-activity-modifying protein 2.

Adrenomedullin (AM) levels are elevated in cardiovascular disease, but little is known of the role of specific receptor components. AM acts via the calcitonin receptor-like receptor (CLR) interacting with a receptor-activity-modifying protein (RAMP). The AM1 receptor is composed of CLR and RAMP2, and the calcitonin gene-related peptide (CGRP) receptor of CLR and RAMP1, as determined by molecular and cell-based analysis. This study examines the relevance of RAMP2 in vivo. Transgenic (TG) mice that overexpress RAMP2 in smooth muscle were generated. The role of RAMP2 in the regulation of blood pressure and in vascular function was investigated. Basal blood pressure, acute angiotensin II-raised blood pressure, and cardiovascular properties were similar in wild-type (WT) and TG mice. However, the hypotensive effect of IV AM, unlike CGRP, was enhanced in TG mice (P<0.05), whereas a negative inotropic action was excluded by left-ventricular pressure-volume analysis. In aorta relaxation studies, TG vessels responded in a more sensitive manner to AM (EC50, 8.0+/-1.5 nmol/L) than WT (EC50, 17.9+/-3.6 nmol/L). These responses were attenuated by the AM receptor antagonist, AM(22-52), such that residual responses were identical in all mice. Remaining relaxations were further inhibited by CGRP receptor antagonists, although neither affected AM responses when given alone. Mesenteric and cutaneous resistance vessels were also more sensitive to AM in TG than WT mice. Thus RAMP2 plays a key role in the sensitivity and potency of AM-induced hypotensive responses via the AM1 receptor, providing evidence that this receptor is a selective target for novel therapeutic approaches.

The influence of vitamin C on systemic markers of endothelial and inflammatory cell activation in smokers and non-smokers.

OBJECTIVE AND DESIGN: To determine the influence of vitamin C supplementation (500 mg, bd, 14 days) on the circulating concentrations of soluble ICAM-1 (a marker of endothelial activation), neopterin (a marker of monocyte activation), and neutrophil elastase (a marker of neutrophil activation) in smokers and non-smokers in a randomised, double-blind, placebo-controlled trial in a hospital setting. SUBJECTS: Twenty smokers (serum cotinine > or = 20 ng ml(-1)) and 20 age- and gender-matched non-smokers (serum cotinine < or = 13.7 ng ml(-1)). RESULTS: At baseline, there was a significant elevation in the concentration of sICAM-1 in smokers (median 247, IQR 199 to 357 ng ml(-1)) compared to non-smokers (median 207, IQR 189 to 227 ng ml(-1); p = 0.014). Vitamin C supplementation did not influence the circulating concentrations of ICAM-1 or neopterin, or leukocyte elastase activity, in smokers, non-smokers, or in the total population. CONCLUSIONS: Markers of monocyte and neutrophil activation were not influenced by smoking status in this study population. However, sICAM-1 concentrations were significantly elevated in tobacco smokers, reflecting tobacco-induced vascular activation that is unaffected by Vitamin C supplementation.

Protein nitration in cutaneous inflammation in the rat: essential role of inducible nitric oxide synthase and polymorphonuclear leukocytes.

1: We have examined the relationship between neutrophil accumulation, NO(*) production and nitrated protein levels in zymosan-mediated inflammation in rat skin in vivo. 2: Rats were anaesthetized and cutaneous inflammation was induced by zymosan (injected intradermally, i.d.). Experiments were carried out up to 48 h, in recovery procedures as appropriate. Assays for neutrophil accumulation (measurement of myeloperoxidase), nitric oxide (assessment of NO(2)(-)/NO(3)(-)) and nitrated proteins (detected by ELISA and Western blot) were performed in skin extracts. 3: The results demonstrate a close temporal relationship between these parameters. Samples were assayed at 1, 4, 8, 24 and 48 h after i.d. injection of zymosan. The highest levels measured of each parameter (P<0.001 compared with vehicle) were found at 4-8 h, with a reduction towards basal levels by 24 h. 4: Selective depletion of circulating neutrophils with anti-neutrophil antibody abolished neutrophil accumulation and protein nitration. In addition substantially decreased NO levels were found. 5: A selective inducible nitric oxide synthase (iNOS) inhibitor, N-3-aminomethyl-benzyl-acetamidine-dihydrochloride (1400W) also significantly reduced neutrophil levels and NO production and substantially inhibited protein nitration. 6: We conclude that the neutrophil leukocyte plays an essential role in the formation of iNOS-derived NO and nitrated proteins in inflammation, in a time-dependent and reversible manner. The NO-derived iNOS also has a role in stimulating further neutrophil accumulation into skin. This suggests a close mechanistic coupling between neutrophils, NO production and protein nitration.

An objective method for grading ultrasound images of carotid artery plaques.

Plaque composition may improve identification of patients at risk of stroke. A new method of grading ultrasound (US) images to assess plaque composition is described. B-mode US images were obtained from 50 carotid specimens. Image analysis parameters were entered into a discriminant analysis package and compared retrospectively with histology. Discriminant functions were derived and then applied prospectively to image-analysis data obtained from a further 50 plaque specimens. For the prospective analysis, US images were graded according to the relative contribution of calcium, fibrous tissue, haemorrhage and lipid. The accuracy for retrospective classification of calcium was 100%, for fibrous tissue 97%, for lipid 76% and 68% for haemorrhage (kappa = 0.81). Prospective classification showed an overall agreement of 65% (kappa = 0.47). Significant intraplaque haemorrhage was identified with an 81% sensitivity and 83% specificity. The US method described demonstrated improved accuracy compared to previous studies. Further study is required to establish the use of this method for in vivo images and its correlation with patient symptoms.

Cell biotinylation provides a sensitive and effective detection technique for cellular adhesion assays: comparison with existing methods.

A simple, sensitive, colorimetric labelling method was devised to quantify cell adhesion, based on labelling the cell plasma membrane with biotin. This method was applied in adhesion assays, which involved the adherence of biotin-labelled, PMA-stimulated, U937 cells. These cells resemble monocytes, and were bound onto fibronectin-coated wells and to an ECV304 cell monolayer. The adherent U937 cells were detected by the addition of a peroxidase-conjugated anti-biotin antibody and a soluble colorimetric substrate. This assay is convenient, fast and sensitive, and able to detect 320-1000 U937 cells under the conditions described. This study has used titration assays to compare the biotinylation method with the existing cell quantification approaches of 51Cr radiolabelling and antibody dependent ELISA. Chromium labelling was the most sensitive technique, but we found the biotinylation method to be more convenient than radioactive labelling and more sensitive than conventional ELISA. Biotinylated cells were also used very effectively in a Stamper-Woodruff adhesion assay with U937 cells binding to histological sections of atherosclerotic plaques. The selective detection of the bound cells permitted automated quantitation by image analysis. Whole cell biotinylation may have wider applications in biological research.

Elective removal of an intramyocardial bullet.

A 26-year-old man had a gunshot wound in the right posterolateral aspect of the chest. A chest radiograph showed the bullet in the region of the cardiac silhouette. The patient was hemodynamically stable and had no complaints of dyspnea or abdominal pain. Echocardiography and computed tomography identified the bullet in the wall of the right ventricle. The surgical management of the injury is discussed in detail.

Increased ICAM-1 and PECAM-1 transcription levels in the heart of Apo-E deficient mice in comparison to wild type (C57BL6).

Adhesion molecules and chemoattractants are thought to play a critical role in the homing of leukocytes to sites of vascular lesions. Apo-E deficiency in mice creates an atherosclerotic model that mimics vascular lesions in man. Little is known on the effect of Apo-E deficiency on expression of adhesion molecules in the hearts of these animals. In this study, male C57BL6 and Apo-E deficient mice were fed a chow diet over periods of time (0 to 20 weeks). The transcription levels of major adhesion molecules (ICAM-1, PECAM-1), present in the heart, were followed by northern blots. Immunohistochemistry was used to localize these adhesion molecules in the heart. Results show a significant increase in gene transcription levels of ICAM-1 and PECAM-1 in Apo-E animals, but not wild type, at 16 and 20 weeks of chow diet. Such increase in levels of transcription was not observed in younger Apo-E and C57BL6 animals (0, 6 weeks of diet). ICAM-1 and PECAM-1 were strongly expressed in the endocardium and heart microvessels. In contrast, VCAM-1 was poorly stained, with only an occasional expression on the endocardium and arterioles. Enhanced gene expression levels of heart ICAM-1 and PECAM-1 observed in Apo-E deficient mice, but not in control animals, appears to induce the initial stages of an inflammatory reaction. Such observations, not previously reported, may induce heart vascular remodeling.

ICAM-1 deficiency reduces atherosclerotic lesions in double-knockout mice (ApoE(-/-)/ICAM-1(-/-)) fed a fat or a chow diet.

Intercellular adhesion molecule (ICAM)-1, a major adhesion molecule, plays a critical role in the homing of leukocytes to sites of atherosclerotic lesions. However, very little is known on the role of ICAM-1 in initiating and perpetuating vascular lesions in ApoE(-/-) mice fed a chow or a fat diet. This study has investigated the mean aortic lesions in mice (C57BL6 background) with a single-knockout (ApoE(-/-)) or double-knockout (DKO; ApoE(-/-), ICAM-1(-/-)) fed a chow or a fat diet over a period of 3, 6, 15, and 20 weeks. A 3-fold reduction in lesion size was observed at all time points in DKO mice fed a chow diet. However, in DKO mice fed a fat diet, a marked reduction in the aortic lesion was observed at 3 and 15 weeks, which did not reach a significant level at 6 and 20 weeks. This study shows in essence that DKO mice are protected from developing significant lesions for up to 6 weeks when fed a chow diet and from 3 to 6 weeks when fed a fat diet. After 6 weeks, the lesion size of the DKO mice follows that of the single-knockout mice when fed a chow diet and gets to the same level in mice fed a fat diet. Plasma cholesterol levels were not altered as a result of ICAM-1 deficiency. These studies show that ICAM-1 is implicated in the formation and progression of atherosclerotic lesions.

Plasma concentrations of reputed tumor-associated soluble CD44 isoforms (v5 and v6) in smokers are dose related and decline on smoking cessation.

There is some evidence to suggest that smoking may affect circulating levels of CD44 (sCD44) molecules. Therefore, we investigated the effect of smoking on the circulating level of sCD44 by comparing the change in total sCD44, sCD44v5, and sCD44v6 concentrations over 1 year in a group of people who quit smoking (n = 30) and a control group of people who continued to smoke (n = 30). Smoking status and compliance were monitored by analysis of plasma cotinine and expired CO levels and also by self-reported tobacco use. We show a dose-dependent relationship between smoke intake and baseline plasma concentrations of reputed tumor-associated CD44 variant isoforms (sCD44v5 and sCD44v6) in smokers (n = 60). There was a significant decline in the level of both sCD44v5 and sCD44v6 in quitters as compared with continuing smokers [-13.2 (95% confidence interval, -7.6 to -18.8; P < 0.001) and -62.2 ng/ml (95% confidence interval, -33.9 to -90.6; P < 0.001), respectively], but not in the total sCD44 concentration. These results show that the increased concentrations of sCD44v5 and sCD44v6 in smokers are dose related and reversible and suggest that the attributed diagnostic specificity and prognostic value of sCD44 molecules in malignant and inflammatory disease may be affected by smoking status.

Ex vivo antisense oligonucleotides to proliferating cell nuclear antigen and Cdc2 kinase inhibit graft coronary artery disease.

BACKGROUND: The long-term success of cardiac transplantation is limited by graft coronary artery disease (GCAD). Antisense oligonucleotides (ASs) to proliferating cell nuclear antigen (PCNA) and Cdc2 kinase (Cdc2 k) can arrest cell cycle progression and inhibit neointimal hyperplasia. Transforming growth factor-ss(1) (TGF-ss(1)) has been implicated in vascular smooth muscle cell (VSMC) activation. The role of TGF-ss(1) in GCAD remains unclear. We hypothesized that ASs to PCNA and Cdc2 k would inhibit VSMC proliferation and GCAD. METHODS AND RESULTS: In vitro VSMC proliferation was determined after pretreatment with AS solution or medium alone followed by angiotensin II stimulation. PVG-to-ACI rat heterotopic cardiac transplantation procedures were performed after ex vivo pressure-mediated transfection of ASs to PCNA and Cdc2k or saline alone. At postoperative days 30, 60, and 90, allografts were assessed for GCAD, percent neointimal macrophages and VSMCs, and TGF-ss(1) activity. AS pretreatment significantly attenuated VSMC proliferation. At postoperative day 90, percent affected arteries, percent occlusion, and intima-media ratio demonstrated severe GCAD in saline-treated allografts, whereas these parameters were significantly lower in AS-treated allografts. Percent neointimal macrophages and VSMCs was reduced in AS-treated allografts. TGF-ss(1) activity was increased in saline compared with AS-treated allografts and nontransplanted heart controls. CONCLUSIONS: ASs to PCNA and Cdc2 k inhibit VSMC proliferation in vitro and reduce GCAD, percent neointimal VSMCs and macrophages, and TGF-ss(1) activity in vivo. TGF-ss(1) may play a "response to injury" role in the development of GCAD. The prevention of GCAD via AS inhibition of cell cycle regulatory genes before reperfusion may offer a useful clinical alternative to current therapeutic strategies.

Modulation of expression of endothelial intercellular adhesion molecule-1, platelet-endothelial cell adhesion molecule-1, and vascular cell adhesion molecule-1 in aortic arch lesions of apolipoprotein E-deficient compared with wild-type mice.

Human vascular adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), platelet-endothelial cell adhesion molecule-1 (PECAM-1), and vascular cell adhesion molecule-1 (VCAM-1), are thought to play a critical role in the homing of leukocytes to sites of atherosclerotic lesions. However, very little is known about the expression of adhesion molecules in the vasculature of mice models, such as apolipoprotein E knockout (apoE(-/-)) mice, the lesions of which closely mimic human atherosclerotic lesions. This study has first quantitatively characterized the mean expression of endothelial adhesion molecules, lining the whole vessel intimal circumference, over a period of time (0 to 20 weeks of diet) in aortic arch lesions of male apoE-deficient compared with wild-type (C57BL/6) mice. These animals were fed a chow or a cholesterol-rich diet. ApoE(-/-) animals showed first an increase (at 6 weeks) and then a reduction (at 16 weeks) in the mean expression of ICAM-1 (P<0.05) and PECAM-1 (P<0.05) but not VCAM-1 levels. Such modulation of the mean expression of adhesion molecules was not observed in wild-type mice. Confirmation of immunohistochemistry results on ICAM-1 was obtained by Northern blots performed on the aortic arch of apoE and C57BL6 chow-fed mice over a period of 20 weeks. Moreover, the presence of VCAM-1 was also confirmed at the RNA level, on aortas of control and apoE mice, by reverse transcription-polymerase chain reaction. In the second part of the study, we assayed the levels of adhesion molecules, in different types of histologically defined atherosclerotic lesions, in apoE(-/-) animals fed for 20 weeks. All 3 adhesion molecules (ICAM-1, PECAM-1, and VCAM-1) were observed to be reduced in fibrofatty and complex lesions but not in fatty streaks or in areas without lesions. These results indicate that the expression of these adhesion molecules in apoE-deficient animals varies with the evolution of the plaque from a fatty to a fibrous stage.

Effects of humanized monoclonal antibody to rhesus CD11a in rhesus monkey cardiac allograft recipients.

INTRODUCTION: Leukocyte function-associated antigen-1 (LFA-1, CD11a) monoclonal antibody (mAb) affects many leukocyte functions without cell depletion. We hypothesized that the use of a humanized, anti-rhesus modified LFA-1 mAb (H2C12) in rhesus monkeys would cause: (1) prolonged heart allograft survival, (2) inhibition of primary but not secondary antibody responses, and (3) minimal drug toxicity. METHODS AND RESULTS: Control (n=5) and H2C12-treated (n=7) (8-20 mg/kg i.v. on day -1 followed by 10 mg/kg/day) adult male rhesus recipients were inoculated with GP120 protein antigen on day -28 and -1 and grafted with heterotopic abdominal hearts (day 0). Donor-recipient pairs were equally MLR mismatched (4329.8+/-1124.1 CPM controls vs. 7289.0+/-1926.5 treated, P=NS). Mean heart allograft survival as evaluated by daily abdominal palpation was significantly prolonged in high dose recipients (23.0+/-2.6, n=4) vesus controls (8.2+/-1.3, n=5, P<0.02, Mann-Whitney U test). H2C12 treatment did not produce signs of cytokine release or toxicity, was nondepleting, but down-modulated PBL CD11a expression to 43.4+/-3.6% (n=4) of control levels (n=5) at day 7 as demonstrated by flow cytometry. It had no effect on postoperative Con A or MLR and did not prevent mAb clearance due to the rhesus-antihuman antibody response. The addition of mycophenolate mofitil prevented rhesus-antihuman antibody response with therapeutic H2C12 levels seen for >35 days. CONCLUSIONS: The use of this mAb to block CD11a had the benefit of being a well tolerated, highly targeted therapy. These are the first results showing that monotherapy with anti-leukocyte function-associated antigen-1 mAb prolonged survival of MLR mismatched allogenic cardiac grafts in primates.

Optimization of ex vivo pressure mediated delivery of antisense oligodeoxynucleotides to ICAM-1 reduces reperfusion injury in rat cardiac allografts.

BACKGROUND: Our purpose was to optimize hyperbaric pressure as a vector for ex vivo transfection of antisense oligodeoxynucleotides (AS-ODN) to intercellular adhesion molecule-1 to limit reperfusion injury (RI) in cardiac allografts. We investigated the effects of increased pressure, incubation time, and AS-ODN concentrations on transfection efficiency and toxicity. METHODS AND RESULTS: PVG (RT1c) donor hearts were heterotopically transplanted to ACI (RT1a) recipients. Donor hearts were harvested and the various groups were treated at: (1) different pressure (1-9 atm) for 45 min with 80 micromol/liter AS-ODN; (2) different incubation times (15 min to 6 hr) at 5 atm with 80 micromol/liter AS-ODN; 3) different AS-ODN concentrations (80-240 micromol/liter) at 5 atm for 45 min. Hearts were procured 24 or 72 hr after transplantation. Transfection efficiency was determined with fluorescein-labeled AS-ODN. The degree of RI was determined with biochemical and histological analysis. Increasing pressure from ambient (1 atm) pressure to pressures as high as 9 atm leads to a increase in transfection efficiency from 1.7+/-.5 to 62+/-3.9% and a reduction in RI. Increased incubation time up to 45 min increased transfection efficiency and reduced RI, but longer incubation times induced significant toxicity to the allograft. Increased AS-ODN concentrations improved transfection and reduced RI. CONCLUSIONS: Hyperbaric pressure is a safe and effective vector for the ex vivo delivery of AS-ICAM-1-ODN to rodent cardiac allografts and results in a reduction in reperfusion injury.

Normal expression of cell adhesion molecules in placentae from women with systemic lupus erythematosus and the antiphospholipid syndrome.

Pregnant women with active systemic lupus erythematosus (SLE) and/or the antiphospholipid syndrome (APS) are prone to recurrent miscarriage, pre-eclampsia, intrauterine growth restriction and premature delivery. Placental dysfunction may account for these complications yet the mechanisms remain uncertain. Amongst these, an inflammatory response in the placental vasculature could play a role, involving recruitment of neutrophils and platelets and the increased endothelial expression of cell adhesion molecules (CAM), central to the recruitment process. The aim of this study was primarily to investigate CAM expression in the fetoplacental vasculature in women with SLE/APS. Circulating maternal concentrations of soluble CAM were also elucidated. There were no differences in CAM immunostaining in placentae from patients with SLE and/or APS compared with controls. In both patients and controls moderate immunostaining for the intercellular adhesion molecule-1 (ICAM-1) was observed in placental vascular endothelium and mild immunostaining was present in the placental villous stroma. Strong immunostaining for platelet endothelial CAM (PECAM) occured in the placental vascular endothelium whereas P-selectin was mildly expressed in the stem vessel endothelium only. Vascular CAM-1 (VCAM-1) and E-selectin were undetectable in either study or control placentae. In contrast, ICAM-1 and VCAM-1 but not E-selectin, as assessed by immunoassay (ELISA), were elevated in maternal serum from SLE/APS patients compared with controls. This study suggests that upregulation of CAM expression and subsequent activation of neutrophil and/or platelet activity within the placental villous tree is unlikely to be a mechanism by which the adverse pregnancy outcome arises in SLE/APS pregnancies. However, maternal endothelial cell activation (ECA) may play a more important role.