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Background— Integrins play an important role for vascular smooth muscle cell (VSMC) migration during the development of atherosclerosis and restenosis. Integrin α v -subunit consists of disulphide-bound 125-kDa heavy and 25-kDa light chains, which are generated by endoproteolytic cleavage. This type of activation requires the presence of suitable proprotein convertases (PCs). Based on ex vivo and in vitro data, the PC5 isozyme has been suggested to be the major integrin convertase. We have recently demonstrated that PC5 is upregulated during vascular remodeling in rodents, colocalizing with α v in VSMCs. The aim of this study was to investigate the activation of α v by PCs in VSMCs and its consequences for α v -dependent cell functions. Methods and Results— Immunoblotting demonstrated that inhibition of PC activity by the specific pharmacological inhibitor dec-CMK inhibits α v cleavage in VSMCs. These results were confirmed using PC5-specific antisense oligonucleotides. PC5-antisense oligonucleotides and dec-CMK inhibited VSMC adhesion to the α v β 3 /β 5 ligand vitronectin (both P P v cleavage inhibited the adhesion-dependent focal adhesion kinase Y397 -autophosphorylation and subsequent Akt activation, whereas phosphorylation of extracellular signal-regulated kinase 1/2 was not affected. In human endarterectomy lesions, PC5 colocalized with α v integrin in VSMCs in the atherosclerotic plaques. Conclusions— The present study demonstrates that α v endoproteolytic activation is necessary for integrin-mediated adhesion and migration as well as signaling and requires PC5 in VSMCs. The colocalization of PC5 and α v in human carotid plaques indicates that PC5 might play a key role for α v activation in vivo.

Background: Mild therapeutic hypothermia (MTH) improves outcome for patients with acute coronary syndrome (ACS) after cardiac arrest (CA). Previous data point to an interaction between hypothermia and drug metabolism, thus potentially impacting on platelet function under antiplatelet therapy. Purpose: The aim of the study is to determine clopidogrel metabolism and platelet function in clopidogrel naïve ACS patients treated with mild hypothermia (33°C, n=12) compared with ACS patients (troponin positive) with normal body temperature (n=14). Methods: Platelet function was measured by light transmittance aggregometry (LTA), multiple electrode platelet aggregometry (MEA) and VASP phosphorylation analysis before, 2h, 4h and 24h after administration of a 600 mg clopidogrel loading dose. Furthermore, plasma concentrations of clopidogrel, the active thiol metabolite and the inactive carboxyl metabolite were determined. All patients were screened for CYP2C19*2 polymorphism and scheduled for PCI. Mild hypothermia was carried out according to current guidelines for 24 hours at a target temperature of 33°C. Results: Plasma concentration of clopidogrel and metabolites were lower in the MTH group after 2h and 4h, respectively (all pex vivo with 33°C and 37°C, respectively. Conclusion: Inhibition of platelet function is decreased under MTH, presumably due to a diminished clopidogrel absorption and metabolization. Thus, these patients might have a higher risk for cardiovascular events despite antiplatelet therapy.

A newly developed fusion protein (L19-IL2) consisting of an anti-ED-B fibronectin single chain antibody and the cytokine interleukin-2 (IL2), is a potent antitumor agent for renal cell cancer. ED-B fibronectin (ED-B) is expressed in inflammatory plaque lesions. Preliminary studies suggested that delivering IL2 locally to atherosclerotic tissue seem to reduce plaque progression. Therefore we investigated the application of L19-Il2 on lesion formation in apoE-deficient mice (apoE −/− ). 6-month old apoE −/− , fed with high fat diet (n = 5/group) were injected intravenously L19-IL2 (4290IU/g bodyweight), with L19 (antibody alone), with the control fusion protein D1-IL2 (4290IU/g bodyweight) or NaCL (control) at day 1, 3 and 5 and were sacrificed at day 7. Plaque lesion formation was analyzed by histology, immunohisto-chemistry (Mac3 = macrophages, CD31; actin, human IL2, ED-B, CD25, CD4), morphometry of the aortic root and by Sudan stain of the thoracic aorta followed by densitometry. Macrophage, ED-B, CD4, CD25 (IL-2 receptor subunit) content was analysed after immunohistochemistry. Results: Treatment with L19-IL2, L19, and D1-IL2 did not affect bodyweight, survival of mice compared to control (NaCL). Serum levels for blood glucose, cholesterol, liver and cardiac enzymes, and troponin were not different between L19-IL2, L19, and NaCL. Cardiac histology was also unaffected. Human IL2 was only detectable in plaque lesions after L19-IL2, but not after L19 or D1-IL2 treatment. L19-IL2 application significantly reduced plaque lesion size in the aortic root (L19-Il2: 23.2 ± 1.7%, L19: 35.2 ± 1.2%, D1-IL2: 31.6 ± 3.0%, NaCL: 31.0 ± 3.2%, p < 0.01), plaque extension in the thoracic aorta (L19-Il2: 5.3 ± 0.7%, L19: 8.2 ± 0.4%, D1-IL2: 9.8 ± 1.0%, NaCL: 8.9 ± 1.2%, p < 0.01). Mac3 immunoreactivity was reduced under L19-IL2, whereas CD4 and CD25 were strongly increased compared to L19, D1-IL2 and NaCL treated groups. Conclusion: A fusion protein, which delivers human IL-2 into plaque lesions, induces T-lymphocyte activation, reduction of macrophages and a reduction in plaque growth. The present study suggests that local application of IL2 activates cellular mechanisms involving T-lymphocytes leading to a reduction of atherosclerotic disease.

Active inflammation has been identified as major criteria for vulnerable atherosclerotic plaques. So far, they are not detectable by conventional diagnostic methods, such as coronary angiography. Therefore, we wanted to investigate whether molecules expressed in inflammatory, unstable plaques, such as CD40 receptor, can be used for molecular imaging and identification of such lesions. Monoclonal antibodies against CD40 were either coupled to cross-linked dextran-coated iron particles (CLIO) for MR imaging, or to the dye Cy5′5 for near-infrared fluorescence (NIRF) imaging. A matched IgG2A isotype antibody was used as negative control. ApoE−/− mice were fed for 4 month with high cholesterol diet and used as a model of atherosclerosis. Histological specimens of aortic tissues showed abundant expression of CD40 in atherosclerotic lesions of these animals. For NIRF imaging, either 25μg or 500μg Cy5′5-CD40 or Cy5′5-IgG2A were injected into the tail vein, and the aorta was imaged 6 and 24 hours after injection. For MR-imaging CLIO-CD40 (25μg/ml protein) or CLIO-IgG2A were injected and MR images were acquired 2 and 6 hours after injection using a 7 T scanner (Bruker). To prove the specificity of this approach NIRF imaging was also performed in double knockout Apo−/− CD40−/− mice High antibody concentrations (500μg Cy5′5-antiCD40) induced high fluorescence in the aortic wall and no difference to Cy5′5-control IgG2A at 6 and at 24 hours was detectable. Low concentrations (25μg) resulted in accumulation of fluorescence in Sudan-positive areas (r2=0.62) whereas low background fluorescence was detected after injection of Cy5′5-IgG2A (p < 0,05, n = 4) and in double knockout Apo−/− CD40−/− mice in the aortic arch. Fluorescence increased slightly after 24 hours compared to 6 hours after injection. Injection of CLIO-anti-CD40 induced a stable signal in the aortic arch in T2 weighted images of ApoE−/− mice compared to control CLIO-IgG2A. Detection of CD40 in atherosclerotic plaques with different imaging modalities is feasible. The approach might serve as a tool for detection of lesions with high inflammatory activity.

Background— Accumulation of macrophages and their in situ expression of matrix metalloproteinases (MMPs) are important determinants of plaque stability. Activation of membrane-bound MT1-MMP, the major activator of pro-MMP-2, requires intracellular endoproteolytic cleavage of its precursor protein. This type of activation typically requires suitable furin-like proprotein convertases (PCs), specifically furin and PC5. The present study was done to investigate the function of MT1-MMP as well as furin-like PCs in mononuclear inflammatory cells. Methods and Results— Macrophage differentiation of human monocytic THP-1 cells was accompanied by increased expression of furin, PC5, and MT1-MMP. Some pro-MMP-2 activation was found in macrophages, but pro-MMP-2 level or activation was not enhanced after stimulation with the proinflammatory mediators tumor necrosis factor-α or lipopolysaccharide. However, culturing of macrophages in conditioned medium from serum-starved vascular smooth muscle cells, which constitutively secrete pro-MMP-2, resulted in a strong pro-MMP-2 activation. Inhibition of furin-like PCs with the specific pharmacological inhibitor decanoyl-RVKR-chloromethylketone (dec-CMK) inhibited MT1-MMP activation in macrophages. Dec-CMK or furin-specific small interfering RNA significantly inhibited macrophage MT1-MMP–dependent activation of vascular smooth muscle cell–derived pro-MMP-2. Flow cytometry demonstrated that human circulating monocytes express furin and PC5, and MT1-MMP and immunohistochemistry revealed their colocalization in macrophages in advanced human atherosclerotic lesions. Conclusions— Furin-like PCs (furin and PC5) play a central role in a MT-MMP–MMP-2 proteolytic cascade, involving provision of macrophage MT1-MMP for the activation of pro-MMP-2 synthesized by other cells. Furin and PC5 are expressed in human peripheral blood mononuclear cells and colocalize with MT1-MMP in macrophages in the atherosclerotic plaque, supporting the hypothesis that they are potential targets in atherosclerosis.

Background—Accumulating evidence suggests that C-reactive protein (CRP), in addition to being a predictor of coronary events, may have direct actions on the vessel wall in the evolution of atherosclerosis. Although accumulation of vascular smooth muscle cells (VSMCs) in the intima is a key event in the development of arterial lesions, apoptosis of VSMCs also plays an important role in progression of atherosclerotic lesions and contributes to increased plaque vulnerability.Methods and Results—In the present study we demonstrate that CRP induces caspase-mediated apoptosis of human coronary VSMCs. DNA microarray analysis was used to identify CRP-regulated genes. The growth arrest– and DNA damage–inducible gene 153 (GADD153) mRNA expression was prominently upregulated by CRP. As confirmed by Northern blot analysis, CRP induced a time- and dose-dependent increase of GADD153 mRNA expression. GADD153, a gene involved in growth arrest and apoptosis in vascular and nonvascular cells, is regulated at both transcriptional and posttranscriptional levels. CRP regulation of GADD153 mRNA expression in VSMCs occurs primarily at the posttranscriptional level by mRNA stabilization. Small interfering RNA (siRNA) specifically targeted to GADD153 reduced CRP-induced apoptosis. GADD153 also specifically colocalized to apoptotic VSMCs in human coronary lesions, further supporting a functional role for GADD153 in CRP-induced cell death.Conclusions—These results demonstrate that GADD153 is a CRP-regulated gene in human VSMCs and plays a causal role in CRP-induced apoptosis. Pharmacological targeting of CRP expression or action may provide a novel therapy for atherosclerosis.

A 55-year-old woman was admitted to our institution because of progressive right heart failure and purulent pericardial effusion. Echocardiography, limited by poor image quality, revealed a small (14 mm in systole), hemodynamically insignificant pericardial effusion. Magnetic resonance cine images showed reduced volumes of both ventricles (left ventricle, 62 mL; right ventricle, 58 mL in end diastole) and normal ejection fraction but paradoxical movement of the atrial and ventricular septum (Movie). The pericardium was thickened (maximum thickness 8 mm), and …

Background Osteopontin (OP) has been identified in cultured rat cardiac fibroblasts, where it contributes to angiotensin II (AII)-induced remodeling processes; in cultured cardiomyocytes; and in macrophages in cardiac tissues with inflammation. However, the presence of OP has not been reported in histological sections of myocardial tissue. In the present study, we investigated (1) the regulation of OP mRNA expression in cultured rat cardiomyocytes; (2) the localization of OP mRNA in neonatal and adult normal and hypertrophied rat hearts; and (3) the histology of OP expression in myocardial specimens from humans either with myocyte hypertrophy or with no pathological changes. Methods and Results Cultured neonatal cardiomyocytes expressed OP mRNA and were immunoreactive for OP. Endothelin-1 (ET-1) and norepinephrine (NE) increased both OP and atrial natriuretic peptide (ANP) mRNA levels twofold to threefold ( P r 2 =.87, P Conclusions The present study provides the first evidence that cardiomyocytes are a prominent source of OP in vivo and suggests that induction of OP expression is strongly associated with ventricular hypertrophy.