Bone marrow- or vessel wall-derived osteoprotegerin is sufficient to reduce atherosclerotic lesion size and vascular calcification.

OBJECTIVE: Osteoprotegerin (OPG) is a decoy receptor for the osteoclast differentiation factor receptor activator of NF-κB ligand. OPG regulates bone homeostasis, and its inactivation in mice results in severe osteoporosis. OPG deficiency in apolipoprotein E (ApoE)(-/-) mice results in increased atherosclerotic lesion size and calcification. Furthermore, receptor activator of NF-κB ligand enhances macrophage-dependent smooth muscle cell calcification in vitro. Here, we hypothesized that reconstitution of ApoE(-/-)OPG(-/-) mice with ApoE(-/-)OPG(+/+) bone marrow (BM) would be sufficient to rescue lesion progression and vascular calcification. Conversely, reconstitution of ApoE(-/-)OPG(+/+) mice with ApoE(-/-)OPG(-/-) BM may accelerate lesion progression and vascular calcification. APPROACH AND RESULTS: ApoE(-/-)OPG(-/-) mice transplanted with ApoE(-/-)OPG(+/+) BM developed smaller atherosclerotic lesions and deposited less calcium in the innominate artery than that of ApoE(-/-)OPG(-/-) mice transplanted with ApoE(-/-)OPG(-/-) BM. There were no differences in lesion size and calcification in ApoE(-/-)OPG(+/+) mice transplanted with BM from ApoE(-/-)OPG(-/-) or ApoE(-/-)OPG(+/+) mice. The large lesions observed in the ApoE(-/-)OPG(-/-) mice transplanted with OPG(-/-) BM were rich in chondrocyte-like cells, collagen, and proteoglycans. Importantly, the ApoE(-/-)OPG(-/-) mice transplanted with OPG(+/+) BM remained osteoporotic, and the ApoE(-/-)OPG(+/+) mice did not show signs of bone loss regardless of the type of BM received. In coculture experiments, macrophages and mesenchymal stem cells derived from ApoE(-/-)OPG(-/-) BM induced more vascular smooth muscle cell calcification than cells derived from ApoE(-/-)OPG(+/+) mice. CONCLUSIONS: These results indicate that OPG derived either from the BM or from the vessel wall is sufficient to slow down lesion progression and vascular calcification independent of bone turnover.

Pathogens and atherosclerosis: update on the potential contribution of multiple infectious organisms to the pathogenesis of atherosclerosis.

It is currently unclear what causes the chronic inflammation within atherosclerotic plaques. One emerging paradigm suggests that infection with bacteria and/or viruses can contribute to the pathogenesis of atherosclerosis either via direct infection of vascular cells or via the indirect effects of cytokines or acute phase proteins induced by infection at non-vascular sites. This paradigm has been supported by multiple epidemiological studies that have established positive associations between the risk of cardiovascular disease morbidity and mortality and markers of infection. It has also been supported by experimental studies showing an acceleration of the development of atherosclerosis following infection of hyperlipidaemic animal models. There are now a large number of different infectious agents that have been linked with an increased risk of cardiovascular disease. These include: Chlamydia pneumoniae, Porphyromonas gingivalis, Helicobacter pylori , influenza A virus, hepatitis C virus, cytomegalovirus, and human immunodeficiency virus. However, there are significant differences in the strength of the data supporting their association with cardiovascular disease pathogenesis. In some cases, the infectious agents are found within the plaques and viable organisms can be isolated suggesting a direct effect. In other cases, the association is entirely based on biomarkers. In the following review, we evaluate the strength of the data for individual or groups of pathogens with regard to atherosclerosis pathogenesis and their potential contribution by direct or indirect mechanisms and discuss whether the established associations are supportive of the infectious disease paradigm. We also discuss the failure of antibiotic trials and the question of persistent infection.

Depletion of resident alveolar macrophages does not prevent Fas-mediated lung injury in mice.

Activation of the Fas/Fas ligand (FasL) system in the lungs results in a form of injury characterized by alveolar epithelial apoptosis and neutrophilic inflammation. Studies in vitro show that Fas activation induces apoptosis in alveolar epithelial cells and cytokine production in alveolar macrophages. The main goal of this study was to determine the contribution of alveolar macrophages to Fas-induced lung inflammation in mice, by depleting alveolar macrophages using clodronate-containing liposomes. Liposomes containing clodronate or PBS were instilled by intratracheal instillation. After 24 h, the mice received intratracheal instillations of the Fas-activating monoclonal antibody Jo2 or an isotype control antibody and were studied 18 h later. The Jo2 MAb induced increases in bronchoalveolar lavage fluid (BALF) total neutrophils, lung caspase-3 activity, and BALF total protein and worsened histological lung injury in the macrophage-depleted mice. Studies in vitro showed that Fas activation induced the release of the cytokine KC in a mouse lung epithelial cell line, MLE-12. These results suggest that the lung inflammatory response to Fas activation is not primarily dependent on resident alveolar macrophages and may instead depend on cytokine release by alveolar epithelial cells.

A 6 week course of azithromycin treatment has no beneficial effect on atherosclerotic lesion development in apolipoprotein E-deficient mice chronically infected with Chlamydia pneumoniae.

OBJECTIVES: To evaluate whether antimicrobial chemotherapy prevents acceleration of atherosclerotic lesion development induced by infection with Chlamydia pneumoniae. METHODS: ApoE-deficient mice which develop hyperlipidaemia and atherosclerosis spontaneously were inoculated intranasally with C. pneumoniae. Animals were treated with azithromycin for 6 weeks after the third inoculation and the atherosclerotic lesion areas in the aortic sinus were measured by computer-assisted morphometry. RESULTS: At 12 weeks post-infection, infected untreated animals developed significantly larger lesion areas compared with sham-inoculated controls (8.7 x 10(4)+/-2.3 x 10(4) microm(2) versus 5.6 x 10(4)+/-2.4 x 10(4) microm(2)). However, there were no differences in lesion size of infected mice treated with azithromycin in comparison with untreated infected controls (11.0 x 10(4)+/-3.0 x 10(4) microm(2) versus 8.7 x 10(4)+/-2.3 x 10(4) microm(2)). CONCLUSIONS: Antibiotic treatment against C. pneumoniae has no beneficial effects on hyperlipidaemia-induced atherosclerosis accelerated by C. pneumoniae in a mouse model.

In vivo and in vitro studies on Anaplasma phagocytophilum infection of the myeloid cells of a patient with chronic myelogenous leukaemia and human granulocytic ehrlichiosis.

AIMS: The occurrence of human granulocytic ehrlichiosis (HGE) in a patient with chronic myelogenous leukaemia (CML) provided an opportunity to study whether Anaplasma phagocytophilum, the aetiological agent of HGE, infects mature or immature cells, both in vivo and in vitro. METHODS: Diagnosis of HGE was confirmed by culture, polymerase chain reaction (PCR), detection of intragranulocytic inclusions, and serology. The infection rates of different myelogenous stages of granulocytic differentiation were determined by microscopy. Anaplasma phagocytophilum infection of the bone marrow was analysed by PCR, culture, and microscopy. In addition, the in vitro growth of A phagocytophilum in the patient's granulocytes and in HL-60 cells (a promyelocytic leukaemia cell line) was compared. RESULTS: Pretreatment blood smears showed that mature granulocytic cells had a higher infection rate with A phagocytophilum than did immature cells. In the original inoculation of the patient's cells into HL-60 cells to isolate A phagocytophilum, the bacterium grew faster in the patient's leukaemic cells than in HL-60 cells. Anaplasma phagocytophilum inclusions were rarely seen in bone marrow granulocytes and PCR was negative. In vitro, two A phagocytophilum isolates grew faster in the patient's granulocytes than in HL-60 cells. CONCLUSIONS: The superior growth in CML cells compared with HL-60 cells suggests that A phagocytophilum preferentially infects mature granulocytes. The higher infection rate of the patient's mature versus immature granulocytes before treatment and the minimal level of infection of the patient's bone marrow support this. It is possible that the primary site of infection in HGE is the peripheral mature granulocytic population.

Development of a lipoprotein based molecular imaging MR contrast agent for the noninvasive detection of early atherosclerotic disease.

INTRODUCTION: Currently there are no clinically available means of noninvasively detecting early atherosclerotic disease because these lesions are characterized by an accumulation of extracellular lipid and foam cells, but a lack of significant wall thickening or architectural distortion. OBJECTIVE: We hypothesize that a paramagnetically labeled low density lipoprotein (LDL) could serve as a functional probe to detect sites of abnormal lipid metabolism in the vessel wall that represent sites of early disease. METHODS: Isolated LDL was first incubated with manganese-mesoporphyrin, a hydrophobic MR contrast agent (MnMeso). Size exclusion chromatography and absorption mass spectroscopy were performed on the resulting samples to prove that an association between the two occurred. Subsequently, foam cell cultures (n=7) were incubated (10-30 microg/ml for 48 h) with these labeled lipoproteins and the T1 relaxivity of centrifuged pellets of these cells was determined by using an inversion recovery sequence on a 1.5T scanner. These results were compared to control measurements made from foam cell cultures fed unlabeled lipoproteins (n=7). RESULTS: Measured T1 relaxation times of the cells fed the MnMeso-LDL (443.3 +/- 51.8 ms) was significantly different from the T1 relaxivity obtained from cells fed unlabeled lipoproteins (661.3 +/- 60.9 ms). These findings indicate that the amount of contrast bound to the constructed lipoproteins is sufficient to produce measurable MR signal changes noninvasively. CONCLUSIONS: The study results support the feasibility of future in vivo MR experiments with labeled lipoproteins to assess lipoprotein kinetics in the vessel wall, which will hopefully provide a means of detecting early atherosclerotic disease.

Laboratory diagnostic techniques for patients with early Lyme disease associated with erythema migrans: a comparison of different techniques.

Recently, a number of refinements in diagnostic modalities for detection of Borrelia burgdorferi infection have been developed. These include large-volume blood cultures, quantitative polymerase chain reaction (PCR) techniques, and 2-stage serologic testing. In the present study, we compared 6 diagnostic modalities in 47 adult patients who had a clinical diagnosis of erythema migrans. Quantitative PCR on skin biopsy-derived material was the most sensitive diagnostic method (80.9%), followed by 2-stage serologic testing of convalescent-phase samples (66.0%), conventional nested PCR (63.8%), skin culture (51.1%), blood culture (44.7%), and serologic testing of acute-phase samples (40.4%). Results of all assays were negative for 3 patients (6.4%). We conclude that the clinical diagnosis of erythema migrans is highly accurate in an area where B. burgdorferi is endemic if it is made by experienced health care personnel, but some patients with this diagnosis may not have B. burgdorferi infection. No single diagnostic modality is suitable for detection of B. burgdorferi in every patient with erythema migrans.

Chlamydia pneumoniae infection accelerates hyperlipidemia induced atherosclerotic lesion development in C57BL/6J mice.

Considerable evidence of an association between Chlamydia pneumoniae infections and cardiovascular disease has emerged. Animal models using genetically altered mice and hypercholesterolemic rabbits have shown a pathogenic role of C. pneumoniae in accelerating atherosclerotic plaque development. In the present study, we evaluated the effect of chronic C. pneumoniae infection on atherosclerosis in C57BL/6J mice, fed either a regular chow diet or a high fat, high cholesterol diet. Infected animals on an atherogenic diet developed significantly larger lesion areas compared with control mice at 18 weeks (2.5-fold increase; 4177+/-777 vs. 1650+/-808 microm(2); P<0.05) and 24 weeks of age (3.3-fold increase; 14139+/-4147 vs. 4298+/-869 microm(2); P<0.02). This study shows that chronic C. pneumoniae infection accelerates atherosclerotic lesion development in diet induced hypercholesterolemic mice, indicating that C. pneumoniae is a co-risk factor of hyperlipidemia in atherogenesis.

Prophylaxis with single-dose doxycycline for the prevention of Lyme disease after an Ixodes scapularis tick bite.

BACKGROUND: It is unclear whether antimicrobial treatment after an Ixodes scapularis tick bite will prevent Lyme disease. METHODS: In an area of New York where Lyme disease is hyperendemic we conducted a randomized, double-blind, placebo-controlled trial of treatment with a single 200-mg dose of doxycycline in 482 subjects who had removed attached I. scapularis ticks from their bodies within the previous 72 hours. At base line, three weeks, and six weeks, subjects were interviewed and examined, and serum antibody tests were performed, along with blood cultures for Borrelia burgdorferi. Entomologists confirmed the species of the ticks and classified them according to sex, stage, and degree of engorgement. RESULTS: Erythema migrans developed at the site of the tick bite in a significantly smaller proportion of the subjects in the doxycycline group than of those in the placebo group (1 of 235 subjects [0.4 percent] vs. 8 of 247 subjects [3.2 percent], P<0.04). The efficacy of treatment was 87 percent (95 percent confidence interval, 25 to 98 percent). Objective extracutaneous signs of Lyme disease did not develop in any subject, and there were no asymptomatic seroconversions. Treatment with doxycycline was associated with more frequent adverse effects (in 30.1 percent of subjects, as compared with 11.1 percent of those assigned to placebo; P<0.001), primarily nausea (15.4 percent vs. 2.6 percent) and vomiting (5.8 percent vs. 1.3 percent). Erythema migrans developed more frequently after untreated bites from nymphal ticks than after bites from adult female ticks (8 of 142 bites [5.6 percent] vs. 0 of 97 bites [0 percent], P=0.02) and particularly after bites from nymphal ticks that were at least partially engorged with blood (8 of 81 bites [9.9 percent], as compared with 0 of 59 bites from unfed, or flat, nymphal ticks [0 percent]; P=0.02). CONCLUSIONS: A single 200-mg dose of doxycycline given within 72 hours after an I. scapularis tick bite can prevent the development of Lyme disease.

Expression of suppressors of cytokine signaling during liver regeneration.

The cytokines TNF and IL-6 play a critical role early in liver regeneration following partial hepatectomy (PH). Since IL-6 activates signal transducers and activators of transcription (STATs), we examined whether the suppressors of cytokine signaling (SOCS) may be involved in terminating IL-6 signaling. We show here that SOCS-3 mRNA is induced 40-fold 2 hours after surgery. SOCS-2 and CIS mRNA are only weakly induced, and SOCS-1 is not detectable. SOCS-3 induction after PH is transient and correlates with a decrease in STAT-3 DNA binding and a loss of tyrosine 705 phosphorylation. This response is markedly reduced in IL-6 knockout (KO) mice. TNF injection induces SOCS-3 mRNA in wild-type mice (albeit weakly compared with the increase observed after PH) but not in TNF receptor 1 or IL-6 KO mice. In contrast, IL-6 injection induces SOCS-3 in these animals, demonstrating a requirement for IL-6 in SOCS-3 induction. IL-6 injection into wild-type mice also induces SOCS-1, -2, and CIS mRNA, in addition to SOCS-3. Together, these results suggest that SOCS-3 may be a key component in downregulating STAT-3 signaling after PH and that SOCS-3 mRNA levels in the regenerating liver are regulated by IL-6.

Human monocyte-derived insulin-like growth factor-2 enhances the infection of human arterial endothelial cells by Chlamydia pneumoniae.

It has been shown that infection of human endothelial cells by Chlamydia pneumoniae is enhanced by co-culturing endothelial cells with human monocytes and is mediated by monocyte-derived soluble factors. This study was conducted to identify the infectivity-enhancing factor. Serum-free conditioned medium of human monocytic cells was fractionated by ultrafiltration. The enhancing activity was found in the fraction in the molecular mass range between 5000 and 10,000 kDa. Recombinant human insulin-like growth factor (IGF)-1 or -2, with a molecular mass of 7500 kDa, was added to the culture medium of human endothelial cells for growing C. pneumoniae. Only IGF-2 enhanced C. pneumoniae growth. Pretreatment of the conditioned medium with a monoclonal antibody against IGF-2 blocked the enhancing activity. This suggests that the infectivity-enhancing factor is IGF-2 and that paracrine interactions between monocytes and endothelial cells in vivo can induce secretory products and sustain infection with C. pneumoniae within atherosclerotic lesions.

Serial measurements of hematologic counts during the active phase of human granulocytic ehrlichiosis.

To describe the changes that occur in blood count parameters during the natural course of human granulocytic ehrlichiosis, we designed a retrospective cross-sectional case study of 144 patients with human granulocytic ehrlichiosis and matched controls who had a different acute febrile illness. Patients from New York State and the upper Midwest were evaluated from June 1990 through December 1998. Routine complete blood counts and manual differential leukocyte counts of peripheral blood were performed on blood samples that were collected during the active illness, and values were recorded until the day of treatment with an active antibiotic drug. Thrombocytopenia was observed more frequently than was leukopenia, and the risk of having ehrlichiosis varied inversely with the granulocyte count and the platelet count. Patients with ehrlichiosis displayed relative and absolute lymphopenia and had a significant increase in band neutrophil counts during the first week of illness. Knowledge of characteristic complete blood count patterns that occur during active ehrlichiosis may help clinicians to identify patients who should be evaluated specifically for ehrlichiosis and who should receive empiric antibiotic treatment with doxycycline.

Antimicrobial susceptibility of Ehrlichia phagocytophila.

Human granulocytic ehrlichiosis is a recently described disease caused by an obligate intracellular gram-negative organism recently named Ehrlichia phagocytophila. To expand our knowledge of the susceptibility of E. phagocytophila, we tested six New York State isolates for susceptibility to 12 antimicrobials using an HL-60 cell culture system. All of the isolates were susceptible to doxycycline (MIC, < or =0.125 microg/ml; minimum bactericidal concentration [MBC], 0.125 to 0.5 microg/ml), rifampin (MIC, < or =0.125 microg/ml; MBC, < or =0.125 microg/ml), ofloxacin (MIC, < or =2 microg/ml; MBC, < or =2 microg/ml), levofloxacin (MIC, < or =1 microg/ml; MBC, < or =1 microg/ml), and trovafloxacin (MIC, < or =0.032 microg/ml; MBC, < or =0.032 microg/ml). Isolates were uniformly resistant to amoxicillin, ceftriaxone, erythromycin, azithromycin, clarithromycin, and amikacin. For one strain, the MBC of chloramphenicol was < or =8 microg/ml. These data suggest that quinolone antibiotics and rifampin may be alternative agents for patients with intolerance to tetracyclines.

Advanced atherosclerotic lesions in the innominate artery of the ApoE knockout mouse.

Most previous studies of atherosclerosis in hyperlipidemic mouse models have focused their investigations on lesions within the aorta or aortic sinus in young animals. None of these studies has demonstrated clinically significant advanced lesions. We previously mapped the distribution of lesions throughout the arterial tree of apolipoprotein E knockout (apoE(-/-)) mice between the ages of 24 and 60 weeks. We found that the innominate artery, a small vessel connecting the aortic arch to the right subclavian and right carotid artery, exhibits a highly consistent rate of lesion progression and develops a narrowed vessel characterized by atrophic media and perivascular inflammation. The present study reports the characteristics of advanced lesions in the innominate artery of apoE(-/-) mice aged 42 to 60 weeks. In animals aged 42 to 54 weeks, there is a very high frequency of intraplaque hemorrhage and a fibrotic conversion of necrotic zones accompanied by loss of the fibrous cap. By 60 weeks of age, the lesions are characterized by the presence of collagen-rich fibrofatty nodules often flanked by lateral xanthomas. The processes underlying these changes in the innominate artery of older apoE(-/-) mice could well be a model for the critical processes leading to the breakdown and healing of the human atherosclerotic plaque.

An overview of the evolution of the atherosclerotic plaque: from fatty streak to plaque rupture and thrombosis.

Today, there is a wealth of information concerning the chronology of the cellular and molecular events associated with the development of cardiovascular diseases. It is now clear that atherosclerosis is largely a result of a dysregulated fibroproliferative inflammatory response [1]. Treatment of cardiovascular disease is rapidly adapting to this new knowledge and the future holds great potential for preventing the disease by blocking the process at multiple stages. This short review provides an overview of the evolution of the atherosclerotic plaque. It focuses on three basic stages of the disease processes: initiation of the fatty streak, transition of the fatty streak to an atheroma, and progression and destabilization of the lesions leading to plaque rupture and occlusive thrombosis. It includes a discussion of the molecular and cellular biology of the atherogenic process with an emphasis on key molecular mediators of the disease process and provides three tables which list examples of some of the key molecular mediators and the stages in which they are purported to play a role.

The good smooth muscle cells in atherosclerosis.

The formation of a fibrous cap made up of intimal smooth muscle cells and connective tissue is part of an attempt by the vessel wall to encapsulate the toxic products accumulating in the necrotic core of atherosclerotic lesions, and should be viewed as a beneficial healing response. In this review, we discuss the development of the intima and the potential origins of the intimal smooth muscle cell with a focus on the unique properties of these cells. We further discuss the role of intimal smooth muscle cells in plaque rupture and in wound healing, and the relationship of wound healing to the loss of lumen that occurs with development of advanced atherosclerotic lesions.

Detection of dissection and remodeling of atherosclerotic lesions in rabbits after balloon angioplasty by magnetic-resonance imaging.

OBJECTIVE: To assess the usefulness of magnetic-resonance imaging (MRI) for monitoring acute changes after angioplasty of preexisting lesions in rabbits with basal lesions similar to those observed in humans. METHODS: A combination of Fogarty balloon injury (1 week after initiation of diet) and a mildly hypercholesterolemic diet (0.2% cholesterol and 5% peanut oil) was used to promote the rapid formation of atherosclerotic lesions in 16 New Zealand white rabbits. After 5 months of the diet, angioplasty was performed on these lesions with a Grüntzig catheter in both iliac arteries and the abdominal aorta. MRI was used to monitor the initial formation of lesions after 3 and 5 months of the diet, and 2 days, 2 weeks, and 1 and 2 months after angioplasty. RESULTS: The combination of early Fogarty injury and mildly hypercholesterolemic diet induced fibroproliferative lesions similar to type Vb atherosclerotic lesions seen in humans. Angioplasty induced deep dissections at the shoulders of lesions in the majority of animals. These dissections often extended into the media. The cellular, proliferative response after angioplasty was localized and limited to sites of dissection. A significant increase in area of arterial wall was observed after angioplasty at sites of dissection without any loss of lumen. In contrast, proximal and distal to the sites of injury, there was no change in wall area but a transient reduction in lumen area. CONCLUSIONS: Comparison of MRI results with histology confirmed that changes in the wall and lumen, including small linear dissections in the lesions and arterial remodeling, are detectable by MRI.

Effect of Chlamydia trachomatis infection on atherosclerosis in apolipoprotein E-deficient mice.

We have previously demonstrated that Chlamydia pneumoniae accelerates plaque formation in apolipoprotein E-deficient (ApoE(-/-)) mice following intranasal inoculations. In this study, we evaluated the effect of respiratory tract infection with Chlamydia trachomatis on the progression of atherosclerosis in ApoE(-/-) mice. The study showed that in contrast to infection with Chlamydia pneumoniae, infection of the lung and aorta with C. trachomatis was mild and transient and did not significantly accelerate plaque development.

Chlamydia pneumoniae induces inflammatory changes in the heart and aorta of normocholesterolemic C57BL/6J mice.

Chlamydia pneumoniae infection induces inflammatory changes in blood vessels in normocholesterolemic rabbits, but it is not known whether the same phenomenon occurs in other animal models. Thus, in this study, C57BL/6J mice were inoculated with C. pneumoniae. Inflammatory changes in the heart or aorta were observed in a small number of chronically infected mice. No evidence of atherosclerotic lesions was found in any of the mice. These findings suggest that chronic C. pneumoniae infection can induce inflammatory changes in the heart and aorta of C57BL/6J mice, but does not initiate definitive atherosclerosis.

Survival of the human granulocytic ehrlichiosis agent under refrigeration conditions.

The human granulocytic ehrlichiosis (HGE) agent in infected blood specimens remained viable during refrigeration at 4 degrees C for up to 18 days. These findings suggest that blood specimens submitted for culture may withstand transportation to a remote laboratory. HGE should be added to the list of infections potentially transmitted by blood transfusion.