A novel role of bone morphogenetic protein-7 in the regulation of adhesion and migration of human monocytic cells.

BACKGROUND: Bone morphogenetic protein (BMP) 7 is abundant in atherosclerotic plaques and increases monocyte pro-coagulant activity by enhancing tissue factor (TF) expression. While several members of the BMP superfamily are able to serve as chemotactic agents for monocytes, the role of BMP-7 in regulation of monocyte motility is not known. AIMS: To assess the effect of BMP-7 on adhesive and migratory properties of human monocytes. METHODS: Chemokinesis, adhesion, and transendothelial migration of BMP-7-treated THP-1 cells and human monocytes were analysed using live-cell imaging, orbital shear, and Boyden chamber assays. Surface presentation of ß2 integrins and phosphorylation status of Akt & focal adhesion kinase (FAK) were studied by flow cytometry and Western blot. RESULTS: High levels of BMP-7 protein were detectable in intimal regions of atherosclerotic plaques; BMP-7 significantly enhanced THP-1 and monocyte chemokinetic properties in vitro (1.21+0.01 and 1.76+0.21 fold increase in crawling distance, respectively). Under orbital shear, adhesion of monocytic cells to microvascular endothelial cell (MVEC) monolayers was also significantly increased by BMP-7 (3.89+1.56 and 2.57+0.97 fold over vehicle). Moreover, BMP-7 accelerated transendothelial migration of THP-1 cells and monocytes towards MCP-1 (5.91+0.88 and 2.96±0.65 fold increase, respectively). BMP-7 enhanced cell surface presentation of ß2 integrins in the active conformation. Observed effects were determined to be Akt and FAK dependent, as shown by pharmacological inhibition. CONCLUSION: BMP-7 directly upregulates adhesion and migration of human monocytic cells via activation of ß2 integrins, Akt, and FAK. Our findings suggest that BMP-7 may serve as a novel contributor to atherogenesis.

Splice variants of tissue factor promote monocyte-endothelial interactions by triggering the expression of cell adhesion molecules via integrin-mediated signaling.

BACKGROUND: TF is highly expressed in cancerous and atherosclerotic lesions. Monocyte recruitment is a hallmark of disease progression in these pathological states. OBJECTIVE: To examine the role of integrin signaling in TF-dependent recruitment of monocytes by endothelial cells. METHODS: The expression of flTF and asTF in cervical cancer and atherosclerotic lesions was examined. Biologic effects of the exposure of primary microvascular endothelial cells (MVEC) to truncated flTF ectodomain (LZ-TF) and recombinant asTF were assessed. RESULTS: flTF and asTF exhibited nearly identical expression patterns in cancer lesions and lipid-rich plaques. Tumor lesions, as well as stromal CD68(+) monocytes/macrophages, expressed both TF forms. Primary MVEC rapidly adhered to asTF and LZ-TF, and this was completely blocked by anti-ß1 integrin antibody. asTF- and LZ-TF-treatment of MVEC promoted adhesion of peripheral blood mononuclear cells (PBMCs) under orbital shear conditions and under laminar flow; asTF-elicited adhesion was more pronounced than that elicited by LZ-TF. Expression profiling and western blotting revealed a broad activation of cell adhesion molecules (CAMs) in MVEC following asTF treatment including E-selectin, ICAM-1 and VCAM-1. In transwell assays, asTF potentiated PMBC migration through MVEC monolayers by ∼3-fold under MCP-1 gradient. CONCLUSIONS: TF splice variants ligate ß1 integrins on MVEC, which induces the expression of CAMs in MVEC and leads to monocyte adhesion and transendothelial migration. asTF appears more potent than flTF in eliciting these effects. Our findings underscore the pathophysiologic significance of non-proteolytic, integrin-mediated signaling by the two naturally occurring TF variants in cancer and atherosclerosis.

Bone morphogenetic protein -7 increases thrombogenicity of lipid-rich atherosclerotic plaques via activation of tissue factor.

Thrombogenicity of atherosclerotic plaques largely depends on plaque morphology. Tissue factor (TF) expression is higher in lipid-rich than in calcified lesions. Although bone morphogenetic protein (BMP) -7 is a known inhibitor of vascular calcification, the role of BMP-7 in the development of plaque thrombogenicity is uncertain. We hypothesized that increased thrombogenic potential of lipid-rich plaques is attributed to activation of TF by BMP-7. We measured levels of BMP-7 and TF proteins in lipid-rich and calcified carotid plaques, and tested the effects of BMP-7 on TF expression in human monocytes in vitro. Quantitative immunohistochemical analysis of endarterectomy specimens for TF and BMP-7 revealed that lipid-rich plaques contained more TF antigen than calcified ones (158.6±25.3 vs 37.4±8.8 AU, p<0.008). Lipid-rich plaques also expressed higher levels of BMP-7 (60.7±5.2 AU) than calcified lesions (31.8±8.6 AU, p<0.021). In vitro treatment of whole blood with BMP-7 markedly increased the population of TF-positive monocytes from 1.5±0.6 % to 31.0±7.6 % (p<0.001). Stimulation of blood with BMP-7 was accompanied by elevated surface presentation of TF antigen in monocytes as TF-dependent fluorescence intensity increased from 5.0±2.6 AU in unstimulated conditions to 15.8±1.9 AU after incubation with BMP-7 (p<0.002). Our data suggest that BMP-7 contributes to increased thrombogenicity of lipid-rich plaques via enhancement of TF expression.

Tumor lysis syndrome occurring after the administration of rituximab for posttransplant lymphoproliferative disorder.

Tumor lysis syndrome (TLS) may occur after the administration of rituximab for lymphoproliferative disorders. We describe the case of a heart transplant recipient who developed TLS after a single dose of rituximab for the treatment of posttransplant lymphoproliferative disorder. Because rituximab is being used more frequently, it is important for transplant physicians to be aware of this potential complication particularly after administering the first dose.

Coronary artery disease is associated with Alzheimer disease neuropathology in APOE4 carriers.

OBJECTIVE: To examine the associations between postmortem Alzheimer disease (AD) neuropathology and autopsy-verified cardiovascular disease. METHODS: The authors examined 99 subjects (mean age at death = 87.6; SD = 8.7) from the Mount Sinai School of Medicine Department of Psychiatry Brain Bank who were devoid of cerebrovascular disease-associated lesions or of non-AD-related neuropathology. Density of neuritic plaques (NPs) and neurofibrillary tangles (NFTs) as well as coronary artery and aortic atherosclerosis, left ventricular wall thickness, and heart weight were measured. Partial correlations were used to assess the associations of the four cardiovascular variables with NPs and NFTs in the hippocampus, entorhinal cortex, and multiple regions of the cerebral cortex after controlling for age at death, sex, dementia severity, body mass index, and ApoE genotype. These analyses were also repeated separately for ApoE4 carriers and noncarriers. RESULTS: The extent of coronary artery disease and to a lesser extent atherosclerosis were significantly associated with the density of cardinal neuropathologic lesions of AD in this autopsy sample (significant correlations between 0.22 and 0.29). These associations were more pronounced for the ApoE4 allele carriers (n = 42; significant correlations between 0.34 and 0.47). CONCLUSIONS: The degree of coronary artery disease is independently associated with the cardinal neuropathological lesions of Alzheimer disease. These associations are primarily attributable to individuals with the ApoE4 allele.

Case report: a 32-year-old woman with familial parangangliomas and acute cardiomyopathy.

We describe the first case of a patient with familial paragangliomas, acute postoperative catecholamine-induced cardiomyopathy, and the subsequent diagnosis of an unsuspected adrenal pheochromocytoma. Relevant literature is reviewed, and treatment options are discussed.

Rapid generation of thrombin by atheroma and platelets.

Rapid re-occlusion of an atheromatous vessel after angioplasty may occur through yet incompletely known mechanisms. Atheromatous plaque has been shown to contain tissue factor (TF) activity. When atheroma extracts (atheroma) and platelets are incubated together a powerful prothrombinase is rapidly generated, which neither platelets nor atheroma alone can generate. Large amounts of thrombin were generated in minutes by many atheroma-platelet mixtures. However in these mixtures, generation of factor (F)Xa activity was not enhanced, but was in fact decreased by platelet tissue factor pathway inhibitor (TFPI) activity. Leukocytes had no appreciable effect in these short-term experiments. Although levels of factor VII and FX in atheroma were extremely low, antibodies to each of these factors inhibited prothrombinase formation. So did an antibody to factor V. A FXa inhibitor, DX 9065a, was very effective in preventing prothrombinase generation. These findings may explain the rapid occlusion that has been observed after angioplasty and point to avenues of prevention.

Refractory humoral cardiac allograft rejection successfully treated with a single dose of rituximab.

Despite improvements in immunosuppression over the last two decades, the risk of allograft rejection is still high in the early postoperative period. Cellular rejection accounts for the majority of these episodes. However, humoral rejection is a distinct phenomenon that carries a high rate of graft loss and mortality. The currently available treatments for this serious clinical problem include anti-lymphocyte antibodies, immune globulin infusions, as well as plasmapheresis, all of which have limitations. We describe a case of refractory humoral cardiac rejection successfully treated with a single dose of rituximab (375 mg/m2). No further episodes occurred with 2 years of follow-up.

Inhibition of tissue factor limits the growth of venous thrombus in the rabbit.

Antibody mediated inhibition of tissue factor (TF) function reduces thrombus size in ex vivo perfusion of human blood over a TF-free surface at venous shear rates suggesting that TF might be involved in the mechanism of deep vein thrombosis. Moreover, TF-bearing monocytes and polymorphonuclear (PMN) leukocytes were identified in human ex vivo formed thrombi and in circulating blood. To understand the role of TF in thrombus growth, we applied a rabbit venous thrombosis model in which a collagen-coated thread was installed within the jugular vein or within a silicon vein shunt. The effect of an inhibitory monoclonal antirabbit TF antibody (AP-1) or Napsagatran, a specific inhibitor of thrombin, was quantified by continuously monitoring 125I-fibrinogen incorporation into the growing thrombi. The antithrombotic effect obtained with the anti-TF antibody was comparable to the effect observed with the thrombin inhibitor napsagatran suggesting that in this animal model the thrombus propagation is highly TF dependent. Immunostaining revealed that TF was mostly associated with leukocytes within the thrombi formed in the jugular vein or in the silicon vein shunt. Ex vivo perfusion experiments over collagen-coated coverslips demonstrated the presence of TF-bearing PMN leukocytes in circulating blood. The results suggest that in rabbits venous thrombus growth is mediated by clot-bound TF and that blocking the TF activity can inhibit thrombus propagation.

Characterization of aortic root atherosclerosis in ApoE knockout mice: high-resolution in vivo and ex vivo MRM with histological correlation.

In vivo, cardiac-gated, black-blood, and ex vivo magnetic resonance microscopy (MRM) images of the aortic root, and histopathology data were obtained from 12 transgenic and wild-type (WT) mice. MRM was performed using a black-blood imaging spin-echo sequence with upstream and downstream in-flow saturation pulses to obtain aortic root images in three contrast techniques: proton density-weighted (PDW), T(1)- (T(1)W), and T(2)-weighted (T(2)W). Aortic wall thickness and area were measured and correlated with histopathology data (R > 0.90). Ex vivo lesion components (lipid core, fibrous tissue, and cell tissue) were identified and characterized by differing image contrast in PDW, T(1)W, and T(2)W MRM, and by histopathology. The differences between WT and transgenic mice for maximal wall thickness and area were statistically significant (P < 0.05). This study demonstrates the feasibility of in vivo murine aortic root lesion assessment and ex vivo plaque characterization by MRM.

Elevating high-density lipoprotein cholesterol in apolipoprotein E-deficient mice remodels advanced atherosclerotic lesions by decreasing macrophage and increasing smooth muscle cell content.

BACKGROUND: HDL cholesterol levels are inversely correlated with coronary heart disease risk in humans, and in animal studies, HDL elevation decreases formation and progression of foam-cell lesions. The potential for HDL to affect preexisting advanced atherosclerotic lesions is not known. To approach this issue, we used a novel mouse aortic transplantation model. METHODS AND RESULTS: ApoE-deficient (EKO) mice were fed a Western-type diet for 6 months, and thoracic aortic segments containing advanced lesions replaced segments of the abdominal aorta of 4-month-old EKO syngeneic mice not expressing (plasma HDL cholesterol approximately 26 mg/dL) or expressing (HDL approximately 64 mg/dL) a human apoAI (hAI) transgene. Both types of recipients had comparable non-HDL cholesterol levels. Five months after transplantation, mice were killed and grafts analyzed. Compared with lesion area in pretransplant mice (0.14+/-0.04 mm(2), mean+/-SEM), there was progression in the EKO recipients (0.39+/-0.06 mm(2), P<0.01). Compared with EKO recipients, hAI/EKO recipients had retarded progression (0.24+/-0.04 mm(2), P<0.05). Immunostaining for CD68 and other macrophage-associated proteins, monocyte chemoattractant protein-1, acyl coenzyme A:cholesterol acyltransferase, and tissue factor, in lesions of pretransplant and EKO recipient mice showed abundant macrophages. In contrast, compared with any other group, lesional macrophage area in hAI/EKO mice decreased >80% (P<0.003), and smooth muscle cell content (alpha-actin staining) increased >300% (P<0.006). The decrease in macrophages and increase in smooth muscle cells was primarily in the superficial subendothelial layer. CONCLUSIONS: Increasing HDL cholesterol levels in EKO mice retards progression of advanced atherosclerotic lesions and remodels them to a more stable-appearing phenotype.

Blood thrombogenicity in type 2 diabetes mellitus patients is associated with glycemic control.

OBJECTIVES: This study was designed to determine whether blood thrombogenicity is related to chronic glycemic control in type 2 diabetes mellitus (T2DM). BACKGROUND: Type 2 diabetes mellitus is associated with accelerated atherosclerosis and a high rate of arterial thrombotic complications. Whether increased blood thrombogenicity is associated with glycemic control has not been properly tested. METHODS: Forty patients with T2DM with hemoglobin A1c (HbA1c) > or =7.5% were selected. Maintaining their current hypoglycemic therapies, patients were randomized into a conservative (diet modification plus placebo) or intensive (diet modification plus troglitazone) hypoglycemic regimen for three months. Blood thrombogenicity was measured at baseline and after three months with the Badimon ex vivo perfusion chamber and assessed as platelet-thrombus formation. The repeated measurements allowed every patient to be his/her own control. RESULTS: Patients in both groups (48% and 74% of the conservative and intensive groups, respectively) improved glucose control (HbA1c reduction > or =0.5%), showing a significant decrease in blood thrombogenicity. A significant positive correlation was observed between the reduction in thrombus formation and the reduction in HbA1c (r = 0.47, p < 0.01). The reduction in HbA1c achieved by both treatments was comparable. Patients without glycemic improvement showed no change in blood thrombogenicity. Improved glycemic control was the only significant predictor of a decrease in blood thrombogenicity. CONCLUSIONS: In T2DM, there is an association between improved glycemic control and blood thrombogenicity reduction. The effect of glycemic control on the thrombotic complications of T2DM patients deserves further investigation.

Dramatic remodeling of advanced atherosclerotic plaques of the apolipoprotein E-deficient mouse in a novel transplantation model.

OBJECTIVE: Regression of atherosclerotic lesions is an important goal. No extensive experimental evidence shows that it can be achieved for advanced lesions. To study this, we developed a model to maintain a long-term change in the plasma lipoprotein environment of advanced arterial lesions of hyperlipidemic (apolipoprotein E [apoE]-deficient) mice. METHODS: The apoE-deficient mice (plasma total cholesterol of 1334 +/- 219 [+/- SEM] mg/dL) on a typical Western diet for 38 weeks had advanced atherosclerotic lesions (ie, beyond the macrophage foam cell stage) throughout the arterial tree. Lesion-containing thoracic aortas were transplanted (replacing a segment of abdominal aorta) into either apoE-deficient or wild-type (WT) (total cholesterol of 86 +/- 10 mg/dL) recipients. Grafts were harvested after 9 weeks. RESULTS: Compared with pretransplant lesions (area = 0.0892 +/- 0.0179 mm(2)), lesion size tended to increase in apoE-deficient to apoE-deficient grafts (0.2411 +/- 0.0636 mm(2); P =.06), whereas a significant reduction was seen in apoE-deficient to WT grafts (0.0214 +/- 0.0049 mm(2); P <.001). Also, foam cells were absent in apoE-deficient to WT grafts, but abundant in pretransplant lesions and apoE-deficient to apoE-deficient grafts. Grafts were evaluated noninvasively in vivo with magnetic resonance imaging, and wall thickening was detected in the apoE-deficient to apoE-deficient group. CONCLUSIONS: Nearly complete regression of advanced atherosclerotic lesions can be achieved with sustained normalization of the plasma lipoprotein profile. Syngeneic arterial transplantation in mice is a novel and valuable model system for atherosclerosis research; and magnetic resonance imaging can detect differences in characteristics in lesions undergoing regression.

Aortic Slimgraft: ex vivo and in vivo study.

In this article we describe a method for assembling an endovascular graft within the aorta with a graft and then a stent introduced sequentially over a guidewire as separate components. In the ex vivo study, an endovascular graft that was 25 mm in diameter was introduced through a 9F introducer. In the in vivo study, smaller endovascular grafts were placed in the aortas of four healthy pigs and five pigs with aortic aneurysms. Our data suggest that this very low profile system may have significant clinical implications because it converts aortic repair into a percutaneous procedure.

Effect of p27 deficiency and rapamycin on intimal hyperplasia: in vivo and in vitro studies using a p27 knockout mouse model.

SUMMARY: Rapamycin, an immunosuppressant and antiproliferative agent, reduces intimal hyperplasia after arterial injury in animal models and in a preliminary study in humans. Rapamycin treatment reportedly increases expression of p27, a cyclin-dependent kinase inhibitor. This mechanism was tested using a p27-deficient (p27 -/-) murine model. Aortic smooth muscle cells from wild-type (WT) and p27 -/- mice were isolated and cultured. Cell proliferation, assessed by cell count and (3)H-thymidine incorporation, was inhibited significantly by rapamycin in WT and p27 -/- cells at concentrations of 1 ng/ml, 10 ng/ml, and 100 ng/ml (p < 0.05, versus control). The in vivo effect on intimal hyperplasia was studied in p27 -/- and WT mice after femoral artery transluminal injury. Rapamycin treatment was started 2 days before injury and maintained for 2 weeks (1 mg/kg per 48 hours, ip). No significant differences in intima-to-media ratio were found between WT (1.1 +/- 0.1) and p27 -/- mice (1.0 +/- 0.1) 4 weeks after injury. Rapamycin significantly (p < 0.05) reduced intima-to-media ratios in both WT (0.7 +/- 0.1) and p27 -/- mice (0.5 +/- 0.1), compared with untreated mice. p27 deficiency did not alter the arterial wall proliferative response to injury. The inhibitory effect of rapamycin on intimal hyperplasia occurred via a p27-independent mechanism. The in vitro data showed that this effect was mediated through decreased proliferation and enhanced apoptosis.

Beta(3)-integrin-deficient mice but not P-selectin-deficient mice develop intimal hyperplasia after vascular injury: correlation with leukocyte recruitment to adherent platelets 1 hour after injury.

BACKGROUND: Intimal hyperplasia contributes to restenosis after percutaneous vascular interventions. Both beta(3)-integrins, alpha(V)beta(3) and alpha(IIb)beta(3) (glycoprotein IIb/IIIa), and leukocytes have been implicated in neointimal formation, based in part on the results obtained using antagonists to 1 or both receptors in animal models. METHODS AND RESULTS: The responses in wild-type mice, beta(3)-integrin-deficient mice, and P-selectin-deficient mice were studied in a model of transluminal endothelial injury of the femoral artery. At 4 weeks, beta(3)-integrin-deficient mice were not protected from developing intimal hyperplasia, whereas P-selectin-deficient mice were protected. Within 1 hour of injury, several layers of platelets deposited on the arteries of wild-type mice and a single layer of platelets deposited on the vessels of beta(3)-integrin-deficient mice; in both cases, leukocytes were recruited to the platelet layer. In P-selectin-deficient mice, the platelet layer was less compact and extended further into the lumen but did not recruit leukocytes. CONCLUSIONS: In a model of transluminal arterial injury, absence of early leukocyte recruitment and not deficiency of beta(3)-integrins correlated with a reduction in neointimal formation. Blockade of P-selectins may be an effective therapeutic strategy to decrease restenosis after percutaneous vascular interventions.

Acyl-CoA:cholesterol acyltransferase inhibition reduces atherosclerosis in apolipoprotein E-deficient mice.

BACKGROUND: Acyl-COA:cholesterol acyltransferase (ACAT) converts cholesterol to cholesteryl esters. The form of ACAT in macrophages, ACAT1, contributes to foam cell formation in the arterial wall and the development of atherosclerosis. Recent studies in a mouse model of atherosclerosis (the apolipoprotein E [apoE]-deficient mouse), however, have suggested that complete deficiency of ACAT1 activity is not antiatherogenic, in part because of toxicity resulting from adverse effects on tissue cholesterol homeostasis. We have tested whether partial inhibition of ACAT1 and ACAT2 (expressed in liver and intestine) activities reduces atherosclerosis development in apoE-deficient mice and avoids toxicity. METHODS AND RESULTS: ApoE-deficient mice were maintained for 17 weeks on a Western-type diet without (control) or with the ACAT inhibitor F-1394 (effective against ACAT1 and ACAT2) at doses of either 300 (low) or 900 (high) mg/kg. Intimal lesion area at the aortic sinus in controls was 0.69+/-0.06 mm(2). F-1394 treatment significantly decreased lesional area by 39% (low) or 45% (high). F-1394 treatment also reduced lesional immunostaining for macrophages by 61% (low) or 83% (high). En face analysis showed that surface lipid staining in control aortas was 20.0+/-2.8%; F-1394 treatment reduced this by 46% (low) or 62% (high). There were no obvious signs of systemic or vessel wall toxicity associated with F-1394 treatment. CONCLUSIONS: Partial ACAT inhibition by F-1394 had antiatherogenic effects in apoE-deficient mice that were achieved without obvious toxicity. Partial ACAT inhibition may have therapeutic potential in the clinical treatment of atherosclerosis.