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We have studied human umbilical vein (HUVEC) and bovine aortic endothelial cells (BAEC) for the presence of elements of the kinin-kallikrein system. Kinin generation was measured in homogenates of endothelial cells using a radioimmunoassay with a human bradykinin antibody; it was measured after homogenization and was constant over a time interval of 120 min. Addition of exogenous kallikrein (50 mU) led to a five fold increase in kinin concentrations after 5 min in the homogenates, which declined within 2 h. Pretreatment of BAEC with dextran sulfate (0.1 mg/ml) resulted in a 80% reduction in kinin generation. Staining of endothelial cells using an antiserum against glandular kallikrein showed kallikrein immunoreactivity in all cells. Glandular kallikrein activity was measured in homogenates by a colorimetric method. Activities of 3.5 ± 0.4 mU/106 cells in HUVEC and 7.5 ± 0.8 mU/106 cells in BAEC were detected. These data indicate the presence of all key elements of the kinin-kallikrein system in the vascular endothelium. Thus, our results support the existence of a local kallikrein-kinin system in the vascular wall which may contribute to the regulation of local inflammatory processes and the regulation of vascular tone.

To document utilization of lipid-lowering therapy, attainment of low-density lipoprotein cholesterol target values, and cardiovascular outcomes in patients hospitalized for acute coronary syndrome in Germany. The Dyslipidemia International Study II was a multicenter, observational study of the prevalence of dyslipidemia and lipid target value attainment in patients surviving any acute coronary syndrome event. Among patients on lipid-lowering therapy for ≥3 months, use of lipid-lowering therapy and lipid profiles were assessed at admission and again at 120 ± 15 days after admission (the follow-up time point). Multivariate logistic regression was used to identify variables predictive of low-density lipoprotein cholesterol target value attainment in patients using lipid-lowering therapy. A total of 461 patients hospitalized for acute coronary syndrome were identified, 270 (58.6%) of whom were on lipid-lowering therapy at admission. Among patients on lipid-lowering therapy, 90.7% and 85.9% were receiving statin monotherapy at admission and follow-up, respectively. Mean (SD) low-density lipoprotein cholesterol levels in patients on lipid-lowering therapy were 101 (40) mg/dl and 95 (30) mg/dl at admission and follow-up, respectively. In patients with data at both admission and follow-up (n = 61), low-density lipoprotein cholesterol target value attainment rates were the same (19.7%) at both time points. Smoking was associated with a 77% lower likelihood of attaining the low-density lipoprotein cholesterol target value. Hospitalization for an acute event does not greatly alter lipid management in acute coronary syndrome patients in Germany. Both lipid-lowering therapy doses and rates of low-density lipoprotein cholesterol target value attainment remained essentially the same several months after the event.

Aims The aim of this study was to investigate the use of a drug-coated balloon (DCB) in daily clinical practice and provide further evidence on the safety and efficacy of paclitaxel-coated balloon treatment using urea as an inert excipient. Methods and results Between December 2013 and December 2015, 757 patients treated for coronary lesions with the IN.PACT Falcon balloon were enrolled in this prospective real-world all-comers registry. The primary outcome was the clinically driven target lesion revascularisation (TLR) rate at 12 months. The secondary outcome was major adverse cardiac events (MACE) defined as cardiac death, myocardial infarction, TLR and target vessel revascularisation (TVR). Out of 805 lesions, 43.1% were de novo, and 53.2% drug-eluting stent (DES) or bare metal stent (BMS) in-stent restenosis (ISR). TLR at 12 months was 6.2% and TVR 8.3%. MACE occurred in 9.7% of patients with a composite of cardiac death in 0.8% and myocardial infarction in 2.7% plus TLR/TVR. Subgroup analysis confirmed a TLR rate of 7.5% for ISR (2.1% BMS and 9.5% DES) and 4.9% for de novo lesions. Conclusions The IN.PACT Falcon urea-based paclitaxel-coated balloon is safe and efficient in de novo and ISR lesions with low rates of TLR/TVR. The high proportion of treatment of de novo lesions indicates that a DCB-only strategy is nowadays common.

Aims Safety and efficacy of percutaneous coronary interventions using the Pantera Lux paclitaxel-coated balloon have been demonstrated in the PEPPER first-in-man trial. This prospective, multicentre, clinical registry aims to evaluate its safety and efficacy in an international real-world setting in a larger cohort of patients. Methods and results Between April 2010 and April 2011, 1,064 patients were treated for predominantly diffuse and proliferative in-stent restenosis of bare metal stents (BMS-ISR) and drug-eluting stents (DES-ISR), or for de novo lesions. Clinical device success was obtained in 98.2% of the patients. The study endpoint was major adverse cardiac events (MACE), defined as a composite of all-cause mortality, non-fatal myocardial infarction and clinically driven target vessel revascularisation, and was 8.5% in the overall, 6.0% in the BMS-ISR, 11.5% in the DES-ISR and 7.0% in the de novo population at six months, and 15.1%, 11.6%, 20.6% and 9.4% at 12 months, respectively. Definitive stent thrombosis occurred in 0.4% of the patients within 12 months. Conclusions Safety and efficacy of the Pantera Lux paclitaxel-coated balloon was confirmed in a real-world setting with low major adverse cardiac event rates in patients with in-stent restenosis or de novo lesions. (ClinicalTrials.gov: NCT01081366).

Recent studies indicate that regulatory T cells (Tregs) attenuate murine atherosclerosis. Since interleukin (IL)-2 induces Tregs proliferation, we tested the impact of L19-IL2, a fusion antibody specific to extra-domain B of fibronectin (ED-B) containing an active human IL-2 molecule, in experimental atherosclerosis.L19-IL2 or appropriate controls were given intravenously to 6 month old Western diet-fed apoE(-/-) mice on day 1, 3, and 5. Human IL-2 was detected on day 7 within atherosclerotic plaques of L19-IL2-treated mice, and magnetic resonance imaging of the plaques showed a significant adventitial gadolinium enhancement on day 7 and 13, suggesting microvascular leakage as a result of the pharmacodynamic activity of L19-IL2. Treatment with L19-IL2 significantly reduced the size of pre-established atherosclerotic plaques at the thoracic aorta (Sudan III stained area) and in the aortic root area (microscopic, morphometric analysis) on day 7 as compared to controls (L19, D1.3-IL2, NaCl) as well as compared to baseline (day 0). Tregs markers Foxp3 and CTLA4 were highly increased in plaques after L19-IL2 treatment compared to controls (p0.01), whereas the macrophage marker Mac3 was significantly reduced (p0.03). Co-treatment with IL-2-receptor blocking antibody PC61 abrogated L19-IL2-induced plaque reduction compared with IgG control (p0.03).L19-IL2 delivers functional IL-2 to pre-established atherosclerotic plaques of WD-fed apoE(-/-) mice resulting in significant plaque size reduction mediated by local Tregs.

OBJECTIVE To determine the concentrations of adipocyte fatty acid–binding protein (AFABP) and other adipocytokines in maternal and cord serum of pregnant women with gestational diabetes mellitus (GDM) and of control subjects and to relate them to indexes of insulin sensitivity. RESEARCH DESIGN AND METHODS In 86 control and 98 GDM pregnant women, venous blood was collected before vaginal delivery and arterial blood from cord immediately after delivery. Serum insulin and adipocytokines were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS GDM women had higher prepregnancy BMI, and data were adjusted for it. Maternal serum insulin, insulin-to-glucose ratio, homeostasis model assessment (HOMA), AFABP, and retinol-binding protein 4 (RBP4) were higher and adiponectin was lower in GDM than in control subjects, whereas serum glucose, insulin, insulin-to-glucose ratio, HOMA, nonesterified fatty acids, and RBP4 were higher and glycerol, AFABP, and adiponectin were lower in cord blood serum of GDM than of control subjects. AFABP and adiponectin in cord serum of control subjects were higher than in maternal serum; in GDM women no difference was found for AFABP in cord versus maternal serum, although adiponectin remained higher in cord. Values of leptin in both groups were lower in cord than in maternal serum, and those of RBP4 were lower in only GDM women. CONCLUSIONS It is suggested that fetal tissues are the main source of cord arterial serum AFABP, and in GDM fetuses AFABP values correlate with adiposity markers. A downregulation of adiponectin and upregulation of RBP4 in GDM mothers and their fetuses may be related to their insulin-resistant condition, whereas changes in AFABP do not seem to be related.

Vascular smooth muscle cell (VSMC) invasion is a key element in atherogenesis and restenosis, requiring integrins for adhesion/de-adhesion as well as matrix metalloproteinases (MMPs) for focalized proteolysis. Among the MMP family, pro-MMP-2 is unique in its activation, depending on the formation of a multiprotein complex with MT1-MMP/TIMP-2 at the cell surface, in which integrin alphavbeta3 participates. Integrin alphav and MT1-MMP are synthesized from precursors via furin-dependent cleavage of their pro-peptide. Furin is the prototypical proprotein convertase highly expressed in VSMCs and human atherosclerotic lesions. Its precise role in the tight network involving MMPs/integrins and their coordination and cooperation required for VSMC invasion is unknown. We demonstrate that furin-inhibition with decanoyl-RVKR-chloromethylketone inhibits VSMC invasion in a comparable degree to MMP inhibitors, which reduce the MT1-MMP-MMP-2 proteolytic cascade. Furin-inhibition did not prevent MT1-MMP/MMP-2 maturation. In contrast, it strongly reduced pro-alphav cleavage, but did not lessen its cell membrane expression. However, inhibition of pro-alphav processing via furin-inhibition strongly reduced pro-MMP-2 binding to the cell surface, thereby lessening its full maturation and diminishing the cell surface in situ proteolysis required for invasion. Thus, our data demonstrate a novel mechanism of furin-dependent alphav cleavage that enhances pro-MMP-2 binding and activation at the cell membrane in cooperation with MT1-MMP in primary VSMCs. Processing of alphav by furin contributes to the recruitment of enzymatic energy to the cell surface, thereby providing focalized proteolysis associated with VSMC invasion.

In this work we propose an adapted level set segmentation technique for the recognition of atherosclerotic plaque tissue on magnetic resonance (MR) images. The images are 2D cross-sectional images and show different profiles from ex vivo human vessels with high variability in vessel shape. We used a curvature-based anisotropic diffusion technique to denoise the MR-images. The segmentation technique is subdivided into three level set steps. Hence, the result of every phase serves as constructive knowledge for the next level set step. By analyzing and combining carefully all available channel information during the first and second step we are able to delineate exactly the vessel walls by using and adapting two well-known level set segmentation techniques. The third step controls an enclosing level set which separates the plaque patterns from healthy media tissue. In this step we introduce a local weighting concept to consider intensity information for conspicuous plaque patterns. Furthermore, we propose the introduction of a maximal shrinking distance (MSD) for the third level set in the vessel wall and compare the results of the local weighting algorithm with and without the concept of the MSD. The incorporation of locally weighted intensity information into the level set method allows the algorithm to automatically distinguish plaque from healthy media tissue. The knowledge of the MSD can improve the segmentation results and enables to delineate tissue areas where plaque is most likely. Copyright © 2009 John Wiley & Sons, Ltd.

Mononuclear cells (MNCs) are the primary cell type involved in the pro-inflammatory state of obesity-linked insulin-resistance, and atherosclerosis. Increased serum levels of MMP-9 are reported in insulin-resistant type 2 diabetic patients. Here we demonstrate insulin facilitating human monocytic THP-1 cell chemotaxis via prolonged Erk1/2-dependent induction of MMP-9. In vivo, significantly increased serum levels of MMP-9 were found in obesity-induced hyperinsulinemic C57BL/J6 mice, which were diminished by treatment with the anti-diabetic PPARγ-ligand pioglitazone. In line with this, pioglitazone inhibited Erk1/2-phosphorylation and subsequent insulin-dependent MMP-9 synthesis in THP-1 cells. Thus, insulin increases MMP-9 gelatinolytic activity in monocytic cells, which results in accelerated chemotaxis. Hyperinsulinemia is associated with enhanced MMP-9 serum levels, potentially facilitating monocyte migration to and infiltration of adipose tissue and the arterial wall, thereby contributing to the increased cardiovascular risk in obese, hyperinsulinemic patients.

It has been shown that ED-B fibronectin (ED-B) is a potential target for plaque imaging. The aim of this study was to test a novel modified single chain anti-ED-B antibody (scFv) conjugated for near infrared fluorescence imaging (NIRF) with tetrasulfonated carbocyanine-maleimide (TSC-scFv) and to examine the association of ED-B with the presence of macrophages in a murine model of atherosclerosis. Expression of ED-B was observed in plaque areas in apolipoprotein E-deficient (apoE(-/-)) mice which increased with age and plaque load. Robust imaging was possible after explantation of the aorta and demonstrated a strong NIRF signal intensity in focal aortic and brachiocephalic plaque lesions, whereas no signals were found in undiseased areas. Plaque lesion ED-B was expressed by smooth muscle cell and was closely associated to macrophage infiltrates. Although not expressed by the same cell type, there was a significant correlation (p0.01) between ED-B and macrophage immunoreactivity. In vitro human coronary and mouse smooth muscle cells significantly increased ED-B expression after angiotensin II and TNF-alpha treatment. This study demonstrates that plaque NIRF imaging is feasible with a novel single chain antibody and that ED-B expression is closely associated with inflammation in experimental atherosclerosis.

Target temperature management (TTM) after cardiac arrest (CA) improves outcome in patients with acute coronary syndrome (ACS). Previous data point to an interaction between hypothermia and drug metabolism, potentially impacting on platelet function in patients on antiplatelet therapy.To compare clopidogrel metabolism and platelet function in clopidogrel naïve ACS patients treated with TTM (33°C, n=15) and in ACS patients (troponin positive) without TTM (n=18).Platelet function was measured by multiple electrode platelet aggregometry (MEA), light transmittance aggregometry (LTA) and VASP analysis before and after administration of a 600mg clopidogrel loading dose. Plasma levels of clopidogrel and its metabolites were measured. All patients were screened for CYP2C19*2 polymorphism and scheduled for PCI. TTM was carried out for 24h at a target temperature of 33°C using a computer feedback surface cooling device in cardiac arrest patients.Plasma concentration of clopidogrel and metabolites was lower in the TTM group after 2 and 4h, respectively (all p0.005 vs. controls), and platelet function tests revealed an attenuated response to clopidogrel with respect to baseline platelet activity in the TTM group. This was significant for MEA, LTA and VASP analysis (all p0.05). Moreover, there was no significant difference in genotype and platelet function determined ex vivo at 33 or 37°C, respectively.Inhibition of platelet function is significantly lessened in TTM at 33°C, likely due to reduced clopidogrel absorption. Patients with TTM might thus have a higher risk for further cardiovascular events despite antiplatelet therapy with clopidogrel.

Serum concentrations of soluble L-selectin by far exceed those of other soluble adhesion molecules, and serum soluble L-selectin concentrations are remarkably stable upon prolonged storage. We present evidence for Ca(2+)-dependent binding interactions between human serum amyloid P (SAP), a proteolysis-resistant pentraxin glycoprotein, and L-selectin, as shown by surface plasmon resonance measurements, protein band shift assays in a native PAGE system, and after SDS-PAGE and membrane transfer. Monoclonal antibodies to L-selectin strongly reduced binding of biotinylated SAP to L-selectin-IgG chimeras immobilized on microtiter plates. As binding was reduced by prior glycopeptidase F treatment of L-selectin but not of SAP, it appears to be based on SAP lectin domain interactions with N-linked L-selectin carbohydrates. In freshly prepared human lymphocytes, SAP incubation induced expression of a beta2 integrin neoepitope associated with high-affinity binding. This was partially blocked by pre-incubation with Fab fragments of two anti-L-selectin antibodies. In flow chamber experiments, SAP inhibited the adherence of human neutrophils to activated endothelium under shear stress. Thus, SAP binds to human L-selectin and affects L-selectin-dependent leukocyte-endothelial interactions.

Objective: Angiotensin II (AII) promotes cardiac fibrosis by increased extracellular matrix production and enhanced interaction of matrix proteins with their cellular receptors, including integrins. AII and other growth factors augment h1-integrin-dependent adhesion and spreading by binside-out signalingQ without affecting the total number of integrin receptors. bInside-out signalingQ involves specific signaling pathways, including protein kinase C (PKC), leading to activation of h1-integrins. In the present study we investigated the mechanisms involved in AII-increased adhesion of adult rat cardiac fibroblasts (CFBs), obtained from Sprague–Dawley rats, to collagen I mediated by h1-integrin. Methods and results: Treatment of CFBs with AII induced a concentration-dependent increase in adhesion to collagen I (2.2-fold, pb0.01) within 3–6 h of treatment. This effect was mediated by h1-integrin via the angiotensin AT1 receptor and was significantly reduced by protein kinase C inhibition. AII significantly induced phosphorylation of PKC epsilon (PKCq), which is involved in h1-integrin-dependent adhesion and motility, and phosphorylation of the cytoplasmatic tail of h1-integrin at threonine 788/789, required for adhesion. Phosphorylation of h1integrin and an increase in adhesion was also induced by l-a-phosphatidylinositol-3,4,5-triphosphate (l-a-PIP3), an activator of endogeneous PKCq. AII failed to increase adhesion in myofibroblasts obtained from PKCq (/) mice, but not in cells obtained from control mice. Coimmunoprecipitation and double immunofluorescence demonstrated that AII induced a close association of PKCq with h1-integrin in CFBs. Conclusion: The present study demonstrates that AII increased h1-integrin-dependent adhesion to collagen I in cardiac fibroblasts by insideout signaling via PKCq and phosphorylation of the cytoplasmatic tail of the h1-integrin. D 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Integrins are heterodimeric alpha/beta receptors that link the cytoskeleton with the extracellular matrix, thereby regulating several cell functions important in atherosclerosis. In vitro, the subtilisin/kexin-like proprotein convertases (PCs), namely PC5 and furin, have been shown to be responsible for the endoproteolytic activation of the alpha(v) integrin subunit. Based on their cleavage activity, these PCs are potential targets in atherosclerosis. In the present study, we investigated the localization of furin and PC5 in different stages of human atherosclerosis. Immunohistochemical analysis of furin and PC5 revealed their presence in vascular smooth-muscle cells and endothelial cells in atherosclerotic and non-atherosclerotic lesions. However, in the more advanced lesions, furin and PC5 staining was significantly expressed in macrophages/foam cells. In vitro, THP-1 derived macrophages contained furin and PC5, and maturation of monocytes to macrophages was accompanied by enhanced alpha(v)beta3 cell-surface expression. Inhibition of furin/PC5 with the specific pharmacological furin-like PC-inhibitor dec-CMK inhibited alpha(v) endoproteolytic activation but did not abolish alpha(v)beta3 cell-surface expression. This indicates that furin/PC5 is required for alpha(v) endoproteolytic activation but not for alpha(v) routing and sorting to the cell surface. In conclusion, our study demonstrates that furin and PC5 are significantly expressed in mononuclear cells in advanced human atherosclerotic lesions, where they regulate alpha(v) endoproteolytic activation.

The IGF-1 receptor (IGF-1R) and MT1-MMP are synthesized as larger precursor proproteins, which require endoproteolytic activation by the proprotein convertases (PCs) furin/PC5 to gain full biological activity. The aim of this study was to investigate the contribution of PCs to IGF-1R and/or MT1-MMP activation in vascular smooth muscle cells (VSMCs) as well as VSMC proliferation/migration, which are key elements in vascular remodeling. Furin and PC5 mRNAs and proteins were found in VSMCs. Inhibition of furin-like PCs with the specific pharmacological inhibitor dec-CMK inhibited IGF-1R endoproteolytic activation. Inhibition of IGF-1R maturation abrogated IGF-induced IGF-1R autophosphorylation, PI3-kinase and MAPK induction, as well as VSMC proliferation (p

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.

Activation of MAPK pathways by angiotensin II (Ang II) is important for cardiac fibroblast (CFB) proliferation and migration. Activity of MAP-kinases is closely controlled by a group of dual-specific MAP kinase phosphatases (MKPs). Lipopolysaccharides (LPS) and cytokines are elevated in patients with heart failure and may contribute to disease progression. In this study, we investigate the effect of LPS on Ang II-induced CFB function. Pretreatment of CFBs with LPS (1 microg/mL; 30 min) almost completely inhibited Ang II-induced DNA-synthesis and inhibited Ang II directed chemotaxis by more than 80%. Compared to controls, LPS pretreatment significantly reduced phosphorylation levels of ERK1/2- and p38 MAPK and induced MKP-1 levels. Silencing MKP-1 with antisense oligodesoxynucleotides reversed the antimitogenic effect of LPS on Ang II-induced CFB DNA-synthesis and migration. Induction of MKP-1 by LPS was inhibited by the protein kinase C (PKC)-inhibitor calphostin C, but not by the ERK1/2-pathway inhibitor PD98059, suggesting that PKC but not ERK1/2 is required for LPS-mediated MKP-1 induction in CFBs. Our data demonstrate that LPS have direct cellular effects in CFBs through an inhibition of Ang II-induced MAPK activity via PKC-mediated induction of MKP-1. This might be relevant with regard to the decreased MAPK activity and increased levels in MKPs reported during chronic heart failure in humans.

The present study examined non-insulin-treated streptozotocin (STZ)-induced diabetic rats to determine the role of kinins in diabetic nephropathy. Their involvement in the renoprotective effect of the angiotensin-converting enzyme inhibitor (ACEI) ramipril was investigated using the bradykinin (BK) B 2 -receptor antagonist, icatibant (HOE 140), or a combination of the two drugs. Although, none of the treatments prevented the decline of the glomerular filtration rate (GFR) in diabetic rats, ramipril (3 mg/kg/day), but not icatibant (HOE 140; 500 μg/kg/day), prevented proteinuria in these animals. However, the antiproteinuric effect of ramipril was reduced by 45% when combined with icatibant. To explore whether the renal kallikrein–kinin system (KKS) belongs to the underlying mechanisms of these findings, we also determined urinary BK levels, renal kallikrein (KLK) and angiotensin-converting enzyme (ACE) activity as well as renal cortical mRNA levels of neutral endopeptidase 24.11 (NEP) and low-molecular weight (LMW) kininogen. STZ led to a reduction of renal KLK and ACE activity and NEP expression and to a three-fold increase of urinary BK excretion and renal kininogen expression. Icatibant given alone had no effect on these parameters. In contrast, ramipril treatment normalized urinary protein and BK excretion as well as kininogen mRNA expression without affecting NEP mRNA expression or KLK and ACE activity. Our data demonstrate that renal BK is increased in severe STZ-induced diabetes mellitus, but may affect glomerular regulation only to a minor degree under this condition. However, kinins are partly involved in the antiproteinuric action of ACEI at this stage of diabetic nephropathy.

Integrin alphav is involved in intracellular-extracellular signaling important for cytoskeleton alterations and control of cell movement. In vitro experiments indicate that the integrin alphav-subunit undergoes post-translational endoproteolytic cleavage. This type of activation requires the presence of suitable kexin/subtilisin-like proprotein convertases. In vitro experiments have demonstrated that, among several proprotein convertases, PC5A, and to a threefold lesser extent furin, can activate alphav integrin. The biological significance of these in vitro data would be further supported by a coexpression and coordinated regulation of the gene expression of alphav integrin and its activating enzyme PC5 in vivo. In the present study we investigated the regulation of alphav integrin and PC5 following balloon injury in vivo. Comparative immunocytochemistry revealed a coordinated regulation of alphav integrin and PC5 during vascular remodeling in rodents. Integrin alphav was found to be upregulated in PCNA-positive, proliferating vascular smooth muscle cells. Northern blots revealed no significant regulation of furin mRNA, whereas PC5A mRNA increased during vascular remodeling, suggesting that PC5 is the major convertase during neointima formation in vivo. Incubation of vascular smooth muscle cells with the Golgi-disturbing agent brefeldin A inhibited alphav integrin maturation, indicating that endoproteolytic cleavage occurs in the trans-Golgi network, were PC5 is localized. Thus, the present study further supports the concept that activation of alphav integrin occurs in the trans-Golgi network in vascular smooth muscle cells and involves PC5.

HMG-CoA reductase inhibitors have direct vascular effects that contribute to plaque stability. In the current study, the authors demonstrate that the HMG-CoA reductase inhibitors atorvastatin and pravastatin augment the adhesion of human (HSMCs) and rat aortic smooth muscle cells (RASMCs) to collagen I via induction of alpha2beta1-integrin receptors. Atorvastatin (0.1 microM ) increased the adhesion of HSMCs to collagen I up to 2-fold (p < 0.01) and pravastatin (1.0 microM ) up to 1.8-fold (p < 0.01) after treatment of at least 24 h. This increase in adhesion was concentration dependent and was observed for treatment periods from 16 to 72 h. Inhibition of isoprenoid synthesis with mevalonate and geranyl-geraniol prevented the statin-induced effect on human and rat smooth muscle cells. Flow cytometry revealed an increased expression of alpha2- and beta1-integrins after treatment with atorvastatin (0.1 microM ) at 24 and 48 h. Atorvastatin increased levels of beta1-integrin mRNA after 12- and 24-h treatment in HSMCs, which was inhibited by mevalonate. Furthermore, atorvastatin (0.1 microM ) and pravastatin (1.0 microM ) inhibited chemotaxis of HSMCs on collagen I, which was also reversed by mevalonate treatment. In contrast, inhibition of beta1-integrins with a specific antibody nearly doubled (p < 0.01) the rate of chemotaxis. These data indicate that the chemotactic activity in HSMCs is inhibited in part by up-regulation of alpha2beta1-integrin receptors. The current study indicates that HMG-CoA reductase inhibitors increase cell-matrix interaction with collagen I via induction of alpha2beta1-integrins and increased adhesion to collagen I.

The enzyme neutral endopeptidase (NEP; EC 3.4.24.11) cleaves several vasoactive peptides such as the atrial natriuretic peptide (ANP). ANP is a hormone of cardiac origin with diuretic and natriuretic actions. Despite elevated circulating levels of ANP, congestive heart failure (CHF) is characterized by progressive sodium and water retention. In order to elucidate the loss of natriuretic and diuretic properties of ANP in CHF we analyzed activity, protein concentrations, mRNA and immunostaining of NEP in kidneys of different models of severe CHF in the rat.CHF was induced by either aortocaval shunt, aortic banding or myocardial infarction in the rat. All models were defined by increased left ventricular end-diastolic pressure and decreased contractility. The diminished effectiveness of ANP was reflected by reduced cGMP/ANP ratio in animals with shunt or infarction. Renal NEP activity was increased in rats with aortocaval shunt (203 +/- 7%, p < 0.001), aortic banding (184 +/- 11%, p < 0.001) and infarction (149 +/- 10%, p < 0.005). Western blot analysis revealed a significant increase in renal NEP protein content in two models of CHF (shunt: 214 +/- 57%, p < 0.05; infarction: 310 +/- 53 %, p < 0.01). The elevated protein expression was paralleled by a threefold increase in renal NEP-mRNA level in the infarction model. The increased renal NEP protein expression and activity may lead to enhanced degradation of ANP and may contribute to the decreased renal response to ANP in heart failure. Thus, the capacity to counteract sodium and water retention, would be diminished. The increased renal NEP activity may therefore be a hitherto unknown factor in the progression of CHF.

Peroxisome proliferator-activated receptors (PPARs) regulate lipid and glucose metabolism and exert several vascular effects that may provide a dual benefit of these receptors on metabolic disorders and atherosclerotic vascular disease. Endothelial cell migration is a key event in the pathogenesis of atherosclerosis. We therefore investigated the effects of lipid-lowering PPARa-activators (fenofibrate, WY14643) and antidiabetic PPARc-activators (troglitazone, ciglitazone) on this endothelial cell function. Both PPARa- and PPARc-activators significantly inhibited VEGF-induced migration of human umbilical vein endothelial cells (EC) in a concentration-dependent manner. Chemotactic signaling in EC is known to require activation of two signaling pathways: the phosphatidylinositol-3-kinase ðPI3K Þ! Akt- and the ERK1/2 mitogen-activated protein kinase (ERK MAPK) pathway. Using the pharmacological PI3K-inhibitor wortmannin and the ERK MAPK-pathway inhibitor PD98059, we observed a complete inhibition of VEGF-induced EC migration. VEGF-induced Akt phosphorylation was significantly inhibited by both PPARa- and c-activators. In contrast, VEGF-stimulated ERK MAPK-activation was not affected by any of the PPAR-activators, indicating that they inhibit migration either downstream of ERK MAPK or independent from this pathway. These results provide first evidence for the antimigratory effects of PPAR-activators in EC. By inhibiting EC migration PPAR-activators may protect the vasculature from pathological alterations associated with metabolic disorders. 2002 Elsevier Science (USA). All rights reserved.

We have recently demonstrated that furin, PC5, and PC7, members of the subtilisin/kexin-like mammalian proprotein convertases (PCs), are found in rodent aorta. These PCs have been identified to activate several growth factors, adhesion molecules and extracellular matrix compounds by endoproteolytic cleavage. In the present study, we investigated the regulation of PC5 in vascular smooth muscle cells (VSMCs) in vitro and in vivo. Stimulation of rat aortic VSMCs with platelet-derived growth factor (PDGF)-BB (20 ng/mL), angiotensin II (Ang II, 1 μmol/L), or 10% fetal calf serum (FCS) for 48 hours increased DNA synthesis, as assessed by proliferating cell nuclear antigen (PCNA) immunoblotting. PC5 was strongly upregulated by PDGF-BB and 10% FCS (both 8-fold, P s6 -kinase (both P s6 -kinase dependent. Thus, growth factors regulate the proprotein convertase PC5, which may play an important role during VSMC growth.

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.

The aim of this study was to investigate whether transformation of quiescent vascular smooth muscle cells (VSMCs) into proliferating secretory cells is accompanied by an expression of processing enzymes that activate de novo-synthesized growth factors. Three enzymes belonging to the family of the kexin/subtilisin-like mammalian proprotein convertases (PCs), furin, PC5, and PC7, were found to be upregulated after balloon denudation in vivo. To determine their importance in these cell processes, we investigated their gene regulation using a short-term organ culture system. After incubation of rat aorta for 4 and 24 hr in serum-free medium, we demonstrated a significant induction of VSMC proliferation. The affected subset of VSMCs, positive for α-smooth muscle actin, also expressed proliferating cell nuclear antigen (PCNA). Our results revealed a parallel upregulation of furin, PC5, and PC7 in PCNA-immunolabeled cells. As a substrate model for comparison with PCs we used nerve growth factor (NGF). NGF is known to be activated by PCs. As shown by Northern blotting analysis, NGF mRNA concentration was significantly increased in cultured explants. NGF was released into the culture medium. In conclusion, both PCs and NGF are coordinately modulated on induction of VSMC proliferation.

Neointima formation involves tissue expression of matrix proteins and growth factors. The role of αvβ3, but not αvβ5 integrin in vascular cells has been sufficiently investigated. The aim of the present study was to determine and compare the function of αvβ3 and αvβ5 integrins in rat aortic (RASMC) and human coronary vascular smooth muscle cells (HCSMC) and to characterize their expression accompanying neointima formation in vivo. RASMC and HCSMC express αvβ3 and αvβ5 integrin subunits. The αvβ5 integrin predominantly mediated adhesion of RASMCs to vitronectin and spreading on vitronectin via RGD-binding sequences. In contrast, the αvβ3 integrin did not contribute to the adhesion and spreading on fibronectin, vitronectin, gelatin or collagen I coated layers. PDGF-directed migration through gelatin coated membranes involved both αvβ3 and αvβ5 integrins. Selective blocking antibodies for αvβ3 and αvβ5 inhibited migration of RASMC and HCSMC by more than 60% (p < 0.01). Integrin expression was studied in vivo in thoracic aorta of Sprague Dawley rats before and after balloon injury. In situ hybridization demonstrated low signals for αvβ3 and β5 mRNA in uninjuried aorta, which increased significantly at 14 days, localized predominantly in the neointima. Northern analysis of aorta after 14 days of injury also demonstrated an upregulation of αvβ3 and β5 mRNA compared to uninjured aorta. Consistent with the increase in message levels, increased inegrin protein expression was seen in the neointima after 7 and 14 days. This study provides evidence that αvβ3 and αvβ5 are elevated during neointima formation in the rat and indicates a novel role for αvβ5 participating in mechanisms regulating smooth muscle cell migration.

The arterial wall is subject to a continuous process of structural, cellular, and molecular modifications that involve cellular growth processes, apoptosis, cell migration, inflammation, and fibrosis, resulting in changes of wall structure and dimension, as well as contractile and elastic properties. Physiological remodeling is an adaptive response to hemodynamic changes in the sense of repair or adjustment. Cardiovascular (CV) diseases, such as diabetes mellitus (DM) and hypertension, as well as aging, lead to enhancement of vascular maladaptive processes and the formation of atherosclerotic lesions and calcifications. One essential consequence of these maladaptive processes is the change of the arterial wall structure and the loss of the elastic properties of the conduit arteries, which is of growing clinical relevance. This phenomenon is termed “arterial stiffening.” This process leads to increased pulse wave velocities and an increase of systolic and pulse pressures attributable to an alteration in the timing of reflected waves.1 Various studies using a variety of indices have established that arterial stiffness is increased with CV risk factors for atherosclerosis, is higher in women, and increases with age even in the absence of vascular disease or risk factors.2,3 Elevated blood pressure related to peripheral vasoconstriction increases aortic stiffness, which is an independent predictor of primary coronary events in patients with essential hypertension.4,5 The relevance of arterial stiffness, carotid pulse pressure, and augmentation index, as independent predictors for CV events, comes from epidemiological studies. The largest amount of evidence has been given for aortic stiffness, measured through carotid-femoral pulse wave velocity (PWV), as >10 000 subjects have been included in studies.1 DM is associated with a high risk for CV morbidity and mortality, including myocardial infarction, left ventricular (LV) hypertrophy, heart failure (HF), and stroke.6 DM leads to abnormal stiffening of the aorta and the large …

CD40-CD154-mediated cell signalling is impor tant for T-cell function under various physiological and pathophysiological conditions. It has been re cently described to play a role in cellular functions of macrophages (MACs) and vascular smooth-muscle cells (VSMCs) in cardiovascular diseases. In the present study, the expression of CD40 receptor protein and its function was studied in VSMCs ob tained from human coronary arteries. In addition, we studied the expression and distribution of CD40 in atherosclerotic lesions of 43 patients. VSMCs isolated from human coronary arteries and aorta treated with interferon-g (INF-γ) demon strated a upregulation of CD40 compared to low basal expression. CD40 expressing VSMCs treated with CD154 (CD40 ligand) resulted in an increas ed expression of inflammatory proteins, such as vascular-cell adhesion molecule-1 (VCAM-1) and Interleukin-8 (Il-8). Serial immunohistological stainings of human iliac arteries from 43 patients were performed (from lesions according to WHO criteria) and CD40 expression and distribution was analyzed. Human atherosclerotic lesions revealed a significant increase of intimal thickness, number of inflammatory infiltrates, and CD40-positive MACs and VSMCs with progression of the lesions. A significant correlation between intimal thickness and CD40-positive MACs and CD40-positive VS-MCs was observed. Ligation of the cellular CD40 receptors on VSMCs contributes to inflammatory cellular events in the vascular wall. The histological data suggest a direct association of CD40 expres sion in atherosclerotic lesions with plaque develop ment and progression of atherosclerosis.

Troglitazone (TRO) is an oral insulin-sensitizer that has direct effects on the vasculature to inhibit cell growth and migration. In vascular smooth muscle cells (VSMCs), insulin transduces a mitogenic signal that is dependent on the ERK1/2 MAP kinases. We examined the effects of TRO on this pathway and found that it inhibits mitogenic signaling. In quiescent VSMCs, insulin (1 microM) induced a 3.2-fold increase in DNA synthesis. TRO (1-20 microM) inhibited insulin-stimulated DNA synthesis by 72.8% at the maximal concentration. TRO at I and 10 microM had no significant effect on insulin-stimulated ERK1/2 activity. At 20 microM, however, TRO modestly enhanced insulin-stimulated ERK1/2 activity by 1.5-fold. ERKs transduce a mitogenic signal by phosphorylating transcription factors such as Elk-1. which regulate critical growth-response genes. We used GAL-Elk-1 expression plasmids to detect ERK-dependent activation of Elk-1. TRO at 1-20 microM potently inhibited insulin-stimulated, ERK1/2-dependent Elk-1 transcription factor activity. Neither early steps in insulin signaling nor the phosphatidylinositol 3-kinase (PI3K) branch of this pathway were affected by TRO, because it had no effect on IRS-1 phosphorylation, PI3K/IRS-1 association, or Akt phosphorylation. Because TRO is a known ligand for the nuclear transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma), we tested two other ligands for this receptor, rosiglitazone (RSG) and 15-deoxy-delta12,14 prostaglandin J2 (15d-PGJ2). Both also inhibited insulin-induced DNA synthesis. In summary, these data show that TRO inhibits mitogenic signaling by insulin at a point distal of ERK1/2 activation, potentially by a PPARgamma-mediated inhibition of ERK-dependent phosphorylation and activation of nuclear transcription factors that regulate cell growth.

Abstract —We recently demonstrated that α v β 3 integrins are involved in the mechanisms of angiotensin II (Ang II)–induced DNA synthesis and collagen gel contractions in rat cardiac fibroblasts (CFBs), cellular mechanisms that are relevant for cardiac remodeling. The aim of the present study was to elucidate the effect of Ang II and other growth factors on the regulation of the α v β 3 integrins in fibroblasts from neonatal rat hearts. The α v β 3 integrin receptor expression was significantly increased ( P 1 (TGF-β 1 ), and platelet-derived growth factor (PDGF) for 8 and 16 hours. The surface expression of the α v and β 3 integrin subunits was elevated after 32 and 48 hours ( P v β 3 integrin (both P v β 3 integrin but was mainly mediated by an α v β 5 integrin. Relevant in vivo expression of α v β 3 integrin by CFBs was confirmed with in situ hybridization with probes for α v and β 3 mRNA in rat hearts. The present study demonstrates that the expression of α v β 3 integrin is augmented by Ang II, PDGF, and TGF-β 1 in neonatal CFBs. Furthermore, this integrin is involved in the chemotaxis, motility, and adhesion of CFBs. The present findings support the current concept that integrins participate in the control of fibroblast behavior during cardiac remodeling mechanisms.

Glycoprotein IIb/IIIa blockade by abciximab and tirofiban, non-peptidergic inhibitors, leads to sustained clinical benefits in the treatment of acute coronary syndromes. The purpose of this study was to clarify the functional effects of abciximab and tirofiban on vascular vitronectin receptors, αvβ3- and αvβ5-integrins. Integrin expression and 7E3 binding in human umbilical venous endothelial cells, human umbilical venous smooth muscle cells, and human iliac arterial smooth muscle cells were observed in the following intensity: αvβ3 — human umbilical venous endothelial cells>human umbilical venous smooth muscle cells>human iliac arterial smooth muscle cells/αvβ5 — human iliac arterial smooth muscle cells>human umbilical venous smooth muscle cells>human umbilical venous endothelial cells. 7E3 binding correlated with αvβ3-expression in all cell types. Integrin-mediated cell functions were analysed with adhesion and spreading assays on vitronectin. In human umbilical venous endothelial cells, these functions were mediated by αvβ3 and in human iliac arterial smooth muscle cells by αvβ5. In human umbilical venous smooth muscle cells, both vitronectin receptors were involved. Abciximab potently inhibited αvβ3-mediated cell adhesion and spreading. With tirofiban, no significant inhibition of vascular cell functions was observed. The present data demonstrate that vitronectin–cell interactions in vascular cells are mediated via two distinct integrin-receptors, αvβ3 and αvβ5. Abciximab, which solely inhibits αvβ3-mediated cell functions, may be particularly effective in human endothelium and in β3-integrin expressing vascular smooth muscle cells.

Wahrend die zellularen Mechanismen der Pathogenese der Arteriosklerose intensiv untersucht worden sind, ist uber die Mechanismen, die zu einer bevorzugten Lokalisation arteriosklerotischer Lasionen in bestimmten Gefasarealen fuhren, weniger bekannt. Zur Untersuchung dieser Mechanismen wurden Endothelzellen aus menschlichen Koronararterien, einem Gefasbereich, in dem haufig arteriosklerotische Lasionen beobachtet werden, isoliert und kultiviert. Endothelzellen der Mikrozirkulation menschlicher Herzen wurden unter gleichen Bedingungen kultiviert und die Reaktion beider Zellarten verglichen. Inkubation der Zellen mit den in bezug auf die Bildung arteriosklerotischer Plaques besonders pathogenen oxidierten LDL induzierte in makrovaskularen koronaren Endothelzellen eine starkere Zunahme der PAI-1-Aktivitat (182%, p < 0,05) als in mikrovaskularen Zellen (144%, p < 0,05), wahrend nichtoxidierte n-LDL die sezernierte PAI-1-Aktivitat nicht beeinflusten. Nach Stimulation mit Angiotensin II (10-11–10-5M) zeigte sich auf makrovaskularen koronaren Endothelzellen eine starkere Expression des Adhasionsmolekuls E-Selectin als auf mikrovaskularen Endothelzellen. Die Angiotensin-II-induzierte E-Selectin-Expression fuhrte in vitro auch funktionell zu einer erhohten Adhasion von HL60-Zellen unter Flusbedingungen. Dagegen wurde die fur die Leukozytenrekrutierung im Rahmen inflammatorischer Reaktionen wichtige L-Selectin-abhangige Adhasion von Angiotensin II nicht induziert. Stimulation mit TNFα fuhrte uberwiegend an mikrovaskularen Endothelzellen zum Auftreten einer L-Selectin-abhangigen Adhasion. Die unterschiedliche Reaktion mikro- und makrovaskularer kardialer Endothelzellen bietet eine Moglichkeit, zellspezifische Differenzierung und praferentielle Lokalisation arteriosklerotischer Lasionen in makrovaskularen Gefasbereichen einerseits und das Auftreten entzundlicher Reaktionen im Bereich der Mikrozirkulation andererseits zu erklaren.

Insulin-stimulated DNA synthesis, MAP kinase (MAPK) activity and c-fos expression in vascular smooth muscle cells (VSMCs) was blocked by the MAPK inhibitor PD 98059. Regulation of c-fos expression by the transcription factor Elk-1 at the serum response element (SRE) is dependent on its phosphorylation by MAPK. PD 98059 also suppressed insulin-induced Elk-1 transcriptional activity through the SRE. These data show that MAPK plays a critical role in both insulin-mediated growth and Elk-1-dependent induction of c-fos in VSMCs.

Abstract Clinical data suggest a link between the activation of the renin-angiotensin system and cardiovascular ischemic events. Leukocyte accumulation in the vessel wall is a hallmark of early atherosclerosis and plaque progression. E-Selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) are adhesion molecules participating in mediating interactions between leukocytes and endothelial cells and have been found to be expressed in atherosclerotic plaques. We investigated whether angiotensin II, the effector of the renin-angiotensin system, influences the endothelial expression of E-selectin, VCAM-1, and ICAM-1. In coronary endothelial cells derived from explanted human hearts, angiotensin II (10 −11 to 10 −5 mol/L) induced a concentration-dependent increase in E-selectin expression. The effect was measured by cell ELISA and duplex reverse-transcription polymerase chain reaction (RT-PCR) and reached its maximum at 10 −7 mol/L. Angiotensin II induced only a small increase in E-selectin expression in cardiac microvascular endothelial cells. VCAM-1 and ICAM-1 were not affected by angiotensin II stimulation. In addition, the effect of angiotensin II–induced E-selectin expression on leukocyte adhesion was quantified under flow conditions. Angiotensin II (10 −7 mol/L) increased leukocyte adhesion significantly to 67% of the maximal effect by tumor necrosis factor-α at a wall shear stress of 2 dyne/cm 2 . This adhesion was found to be E-selectin dependent, as demonstrated by blocking antibodies. The AT 1 -receptor antagonist DUP 753 significantly reduced E-selectin–dependent adhesion, whereas the AT 2 -receptor antagonist PD 123177 had no inhibitory effect. In addition, only AT 1 -receptor, but not AT 2 -receptor, mRNA could be detected by RT-PCR in coronary endothelial cells. Therefore, it is suggested that AT 1 receptors mediate the effects of angiotensin II on E-selectin expression and leukocyte adhesion on coronary endothelial cells.

The expression level of angiotensin II (ANG II) type 1 receptors (AT1) determines the magnitude of ANG II signaling in vascular smooth muscle cells (VSMC). AT1 mRNA expression in cultured bovine VSMC increased twofold after 8 h of protein kinase C (PKC) activation with phorbol 12-myristate 13-acetate (PMA), whereas stimulation with forskolin did not alter the AT1 mRNA level. The expression of AT1 promoter/exon 1 [-513/+92 base pairs (bp)] luciferase constructs transfected into VSMC increased 2.4-fold with PMA stimulation. In-gel kinase assays demonstrated rapid phosphorylation of mitogen-activating protein kinases (MAPK) ERK1 and ERK2 by PMA. Electrophoretic gel mobility shift assays showed sequence-specific binding of nuclear proteins from PMA-activated VSMC, identified as activator protein 1 (AP-1) complex in competition assays, to a radiolabeled AT1-promoter fragment (-368/-399 bp). Recombinant AP-1 binds in a sequence-specific manner to the -386/-399-bp region. Site-specific mutagenesis destroying the AP-1 site, the adjacent polyoma enhancer activator 3 element, or both sites simultaneously indicated that both sites together are necessary and sufficient to control basal and PMA-induced activation of the human AT1 promoter in transfected VSMC. The capability of the phorbol ester PMA to activate the human AT1 promoter in VSMC via an AP-1 element suggests a prominent role for PKC/MAPK and Ets proteins in AT1 regulation.

Migration of vascular smooth muscle cells (VSMCs) is a crucial response to vascular injury resulting in neointima formation and atherosclerosis. Platelet-derived growth factor (PDGF-BB) functions as a potent chemoattractant for VSMCs and enhances these pathologies in the vasculature. However, little is known about the intracellular pathways that mediate VSMC migration. In the present study, we investigated the role of mitogen-activated protein kinase (MAPK) activation in this function, since PDGF-BB as well as other growth factors activate this pathway. Using an in-gel kinase assay, we observed that PD 98059, an inhibitor of MEK that activates MAP kinase, inhibited PDGF-BB-induced activation of ERK-1 and ERK-2 in cultured rat aortic smooth muscle cells in a concentration-dependent manner. In contrast, PDGF-mediated activation of intracellular calcium release was not affected by PD 98059. The chemotactic response of both rat aortic smooth muscle cells (RASMCs) and human umbilical vein smooth muscle cells (HUSMCs) toward PDGF-BB (10 ng/mL) was significantly reduced by PD 98059 (10 μmol/L) to 41.7±7.1% in RASMCs ( P >.01) and to 47.2 ±5.3% in HUSMCs ( P >.01). Similar inhibition was seen at 30 μmol/L, less at 1 μmol/L. To further confirm the specificity of these results implicating the MAPK pathway, an antisense oligodeoxynucleotide (ODN) directed against the initiation translation site of rat ERK-1 and ERK-2 mRNA was used to suppress MAP kinase synthesis and function in rat VSMCs. Liposomal transfection with 0.4 μmol/L antisense ODN reduced ERK-1 and ERK-2 protein by 65% ( P >.01) after 48 hours. The chemotactic response to PDGF-BB (10 ng/mL) was reduced by 75% ( P >.01) in rat VSMCs transfected with the same antisense ODN concentration. Sense and scrambled control ODNs (0.4 μmol/L) did not affect ERK-1 and ERK-2 protein concentrations or chemotaxis of VSMCs induced by PDGF-BB. These experiments provide the first evidence that activation of MAPK is a critical event in PDGF-mediated signal transduction regulating VSMC migration.

BACKGROUND Impaired response to dual antiplatelet therapy is associated with worse cardiovascular outcome. Besides antiplatelet effects, there is evidence that both clopidogrel and acetylsalicylic acid (ASA) have anti-inflammatory properties. However, little is known about the relationship between platelet function and inflammation under dual antiplatelet therapy in patients with stable coronary artery disease. PURPOSE The purpose of the study was to investigate the correlation of platelet function with soluble (s)P-selectin and soluble (s)CD40L in patients undergoing elective percutaneous coronary intervention. Poor response to ASA and clopidogrel could lead to increased levels of inflammatory markers. METHODS A total of 148 patients were included. Eighty percent of the patients were on 100 mg ASA and all patients were clopidogrel naive. They underwent percutaneous coronary intervention and received a loading dose of 600 mg clopidogrel. Platelet function was assessed by light transmittance aggregometry (LTA) and vasodilator-stimulated phosphoprotein analysis at baseline, 24 h after loading, and after 1 month of maintenance therapy, respectively. Plasma levels of sP-selectin and sCD40L were measured. To classify low responders to clopidogrel, patients were screened for genetic variants determining clopidogrel absorption and metabolization. RESULTS sP-selectin levels correlated with LTA findings after stimulation with arachidonic acid (P=0.012). Further, in addition to decreased platelet reactivity observed on LTA, lower sP-selectin levels were seen in patients under ASA therapy (P=0.004). CYP2C19*2 allele carriers had a higher platelet reactivity after clopidogrel loading measured by adenosine diphosphate-induced aggregation in LTA (P=0.008) and vasodilator-stimulated phosphoprotein phosphorylation (P=0.035); however, there was no difference in the inflammatory markers. Multiple regression analysis showed that variables significantly related to sP-selectin plasma levels were sCD40L (P

Vascular smooth muscle cell (VSMC) proliferation and mi- gration are responses to arterial injury that are highly im- portant to the processes of restenosis and atherosclerosis. In the arterial balloon injury model in the rat, platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) are induced in the vessel wall and regulate these VSMC activities. Novel insulin sensitizing agents, thiazo- lidinediones, have been demonstrated to inhibit insulin and epidermal growth factor-induced growth of VSMCs. We hypothesized that these agents might also inhibit the effect of PDGF and bFGF on cultured VSMCs and intimal hyper- plasia in vivo. Troglitazone (1 m M), a member of the thiazo- lidinedione class, produced a near complete inhibition of both bFGF-induced DNA synthesis as measured by bro- modeoxyuridine incorporation (6.5 6 3.9 vs. 17.6 6 4.3% cells labeled, P , 0.05) and c-fos induction. This effect was asso- ciated with an inhibition (by 73 6 4%, P , 0.01) by troglita- zone of the transactivation of the serum response element, which regulates c-fos expression. Inhibition of c-fos induc- tion by troglitazone appeared to occur via a blockade of the MAP kinase pathway at a point downstream of MAP kinase activation by MAP kinase kinase. At this dose, troglitazone also inhibited PDGF-BB-directed migration of VSMC (by 70 6 6%, P , 0.01). These in vitro effects were operative in vivo. Quantitative image analysis revealed that troglita- zone-treated rats had 62% ( P , 0.001) less neointima/media area ratio 14 d after balloon injury of the aorta compared with injured rats that received no troglitazone. These results suggest troglitazone is a potent inhibitor of VSMC prolifera- tion and migration and, thus, may be a useful agent to pre- vent restenosis and possibly atherosclerosis. ( J. Clin. Invest. 1996. 98:1897-1905.) Key words: restenosisplatelet- derived growth factorbasic fibroblast growth factorc-fos • MAP kinase

Activation of vascular beta-adrenergic receptors prevents an increase in vascular permeability caused by free radicals or inflammatory peptides. Methylxanthines seem to have similar protective effects on vascular endothelium. In the present study we investigated the effect of theophylline on the beta-adrenergic receptor expression and cAMP concentrations in cultured endothelial and smooth muscle cells from bovine aorta. Comparable values for beta-receptor density and binding affinity were detected in both cell types. Isoproterenol induced significant downregulation of beta-receptors in endothelial (BAEC: -60.5%) and smooth muscle cells (BASMC: -52.5%; P0.01). Incubation of endothelial cells with theophylline (4 micrograms/ml and and 40 micrograms/ml) for 24 hours did not affect beta-receptor expression, whereas in smooth muscle cells the beta-receptor density was reduced for -31.5% and -28.7%, respectively. In endothelial cells a transient effect on cAMP concentrations was observed after stimulation with isoproterenol (1 microM), but no effect was found in theophylline treated endothelial cells. Stimulation of intact smooth muscle cells with isoproterenol and theophylline (4 micrograms/ml and 40 micrograms/ml) resulted in a significant increase of cAMP concentrations after 60 and 240 minutes. The present data suggest a novel, celltype specific effect of theophylline on the beta-adrenergic receptor expression in vascular smooth muscle cells in vitro.

Abstract Neutral endopeptidase 24.11, a membrane-bound metallopeptidase, cleaves and degrades vasoactive peptides such as atrial natriuretic peptide, endothelin, angiotensin I, substance P, and bradykinin. Therefore, the presence of this metallopeptidase may contribute to the regulation of vascular tone and local inflammatory responses in the vascular endothelium and elsewhere. We determined neutral endopeptidase in cultured human endothelial cells from different vascular beds and studied its regulation by protein kinase C. Neutral endopeptidase was detected in all cultured endothelial cell types. Lowest concentrations were measured in human endothelial cells from umbilical veins (360±14 pg/mg protein), followed by pulmonary and coronary arteries; higher concentrations were found in endothelial cells from the cardiac microcirculation (1099±73 pg/mg protein). Neutral endopeptidase content increased during cell growth but was not affected by endothelial cell growth factor or modifications of the growth medium. Stimulation of protein kinase C with 1-oleoyl-2-acetyl-rac-glycerol (0.1 to 1 μmol/L) and phorbol 12-myristate 13-acetate (0.01 to 0.1 μmol/L) induced a time- and concentration-dependent increase of endothelial cells that was inhibited by cycloheximide (5 μmol/L), an inhibitor of protein synthesis. Incubation with phospholipase C (1 μmol/L) and thrombin (10 IU/mL) induced upregulation of neutral endopeptidase, resulting in 158±26% and 150±22% increases, respectively, compared with controls. The thrombin effect was inhibited by calphostin C (1 μmol/L), an inhibitor of protein kinase C. Endothelial neutral endopeptidase is constitutively expressed in endothelial cells from different origins and is inducible by thrombin via activation of the protein kinase C pathway.

Endothelial neutral endopeptidase (EC 3.4.24.11, NEP) contributes to the inactivation of vasoactive and inflammatory peptides such as f-Met-Leu-Phe, substance P, atrial natriuretic peptide, and bradykinin. The aim of the present study was to investigate the cellular regulation of NEP expression in human endothelial cells, focusing on the role of cyclic nucleotides and cellular phosphodiesterases (PDE). Activation of adenylate cyclase by forskolin or prostaglandin E 1 (PGE 1 ) induced an increase of NEP activity and NEP protein after 24 h of incubation. This effect was mimicked by two activators of protein kinase A, dibutyryl-cAMP and 8-bromo-cAMP. The nonspecific PDE inhibitor, 3-isobutyl-1-methylxanthine (200 μ M ), increased NEP activity up to 192%. The activator of guanylate cyclase, sodium nitroprusside (SNP), did not affect NEP activity but completely inhibited the 3-isobutyl-1-methylxanthine-mediated increase of NEP activity. The PDE-III inhibitors motapizone (100 μ M ) and enoximone (100 μ M ) enhanced NEP activity up to 188% and 213%, the PDE-IV inhibitor rolipram (3 μ M ) up to 162%, and the combined PDE- III IV inhibitor zardaverine (1 μ M ) up to 176% of control values. The present data provide evidence for a cAMP-mediated increase of NEP activity in human endothelial cells.

In recent years, drug coated balloons have emerged as treatment option for PCI. The present registry aims to evaluate the safety and efficacy of the Pantera Lux Paclitaxel Coated Balloon in a real world setting. Between April 2010 and April 2011, 1'064 patients were enrolled at 62 sites in 12

Contemporary PCI Practices Across Three Continents : A Glimpse Into the Preliminary Results of e-NObori Study

We describe the development of a new ELISA for the detection of neural endopeptidase 3.4.24.11 (NEP). Neutral endopeptidase 3.4.24.11 was determined in preparations of human granulocytes, mononuclear cells (MNC), and in serum. Human recombinant NEP was used as reference. Specificity of the mAbs was tested using APAAP, FACS analysis, and Western blot analysis. Lysis of the blood cells was performed by incubating the cells with 0.4% Tween-20 and repeated freezing cycles. The minimal detectable dose for recombinant NEP was 15 pg/ml. The recovery was 94 +/- 9%. The NEP was detectable in 15 out of 20 serum samples of 20 volunteers (mean +/- SEM, 245 +/- 88 pg/ml, n = 20)) and in all granulocyte preparations (1176 +/- 138 pg/10(7) cells, n = 20)). The results were reproducible among replicates (CV = 3 +/- 1%, n = 40), dilutions (CV = 8 +/- 2%, n = 5), and assays (CV = 12 +/- 4%, n = 5). With this new ELISA, a simple and reproducible method for the measurement of NEP 3.4.24.11 is described.