Jürgen R. Sindermann

Differences in the effects of HMG-CoA reductase inhibitors on proliferation and viability of smooth muscle cells in culture

We investigated the influence of lovastatin, simvastatin and pravastatin on proliferation and viability of vascular smooth muscle cells (SMC) in vitro and studied the effects of lovastatin on a mouse SMC line transgenic for a temperature-sensitive mutant of SV40 large T antigen (TAg), known to inhibit the function of p53 and pRb family members. We found that lovastatin and simvastatin inhibited cell proliferation by provoking G0/G1 phase arrest with concomitant depression of the proliferation antigen Ki-67/MIB-1. Lovastatin at high concentrations of 20 micromol/l caused cell death in the presence of serum but not under serum starved conditions, which was verified on the basis of increased DNA strand breaks, decreased DNA content and morphological alterations seen by electron microscopy. Cell death was also found for simvastatin, whereas pravastatin did not exhibit antiproliferative or cytotoxic effects. Mouse SMC transgenic for TAg did not show any impaired sensitivity to the antiproliferative and cell death inducing effect of lovastatin, but both effects could be antagonized by the supplementation of mevalonate. The data indicate that antiproliferative and cytotoxic effects of lovastatin are caused by the using up of products of mevalonate metabolism and do not require the presence of p53 or pRb.

Comparison of the Prognostic Usefulness of N-Terminal Pro-Brain Natriuretic Peptide in Patients With Heart Failure With Versus Without Chronic Kidney Disease

In patients with chronic heart failure (CHF), N-terminal pro-brain natriuretic peptide (NT-pro-BNP) predicted poor outcome. Clinical predictors of NT-pro-BNP and its usefulness in the presence of chronic kidney disease (CKD) are largely unknown. A total of 341 patients with stable CHF were enrolled, of whom 183 (54%) had CKD. During a follow-up of 620 +/- 353 days, 57 patients (17%) experienced a cardiac event (cardiac death, need for extracorporeal assist device, or urgent cardiac transplantation), and 64 patients (20%) were rehospitalized because of worsening CHF. NT-pro-BNP was related to New York Heart Association functional class (R = 0.44, p <0.001) and inversely related to ejection fraction (R = -0.52, p <0.001) and glomerular filtration rate (R = -0.32, p <0.001). A cardiac event was independently predicted by NT-pro-BNP (hazard ratio [HR] 1.56, p <0.001), ejection fraction (HR 0.95, p = 0.018), and serum sodium (HR 0.89, p = 0.004). Using receiver-operator characteristic analysis, NT-pro-BNP > or =1,474 pg/ml best separated patients with or without cardiac events. In patients without CKD, outcome was significantly worse in patients with NT-pro-BNP >1,474 pg/ml in comparison to patients with NT-pro-BNP <1,474 pg/ml (event-free survival rate 0% vs 75%; p <0.001). In patients with CKD, outcome was also significantly worse in subjects with NT-pro-BNP >1,474 pg/ml in comparison to those with NT-pro-BNP <1,474 pg/ml (event-free survival rate 48% vs 93%; p <0.001). NT-pro-BNP independently predicted rehospitalization caused by worsening CHF (HR 1.26, p = 0.023), and a cut-off value of 1,474 pg/ml also separated patients with poor and intermediate prognosis in the CKD and non-CKD groups. In conclusion, NT-pro-BNP independently predicted morbidity and mortality in patients with CHF with and without CKD.

Clinical and echocardiographic outcomes after implantation of the Trifecta aortic bioprosthesis: an initial single-centre experience.

OBJECTIVES The Trifecta valve (St. Jude Medical) was introduced into clinical practice as a tri-leaflet stented pericardial valve designed for supra-annular placement in the aortic position. The present study aims to evaluate the preliminary results with this new bioprosthesis. METHODS Seventy patients underwent aortic valve replacement (AVR) with the Trifecta valve between August 2010 and December 2011. Thirty-three patients were male and 37 were female (52.9%). Mean age was 74.65 ± 7.63 (range 47-90 years). Prevalent cause of AVR was aortic stenosis in 64 (91.43%) patients. The mean preoperative pressure gradient was 50 ± 17 (range 20-84 mmHg), and the mean aortic valve area was 0.77 ± 0.33. Five (7.14%) patients were operated on due to aortic valve endocarditis. One patient was operated on due to isolated, severe aortic insufficiency. All patients were in New York Heart Association functional class III or IV. Twenty-eight (40%) patients underwent concomitant procedures. RESULTS Concomitant procedures were coronary artery bypass grafting (n = 25), mitral valve replacement (n = 1), ablation of atrial fibrillation (n = 1) and septal myomectomy (n = 1). There were no intraoperative deaths. The 30-day in-hospital mortality was 2.85% (2 of 70). One late death occurred during the in-hospital stay due to a multiorgan failure on postoperative day 60. There were 2 (2.85%) perioperative strokes. Mean pressure gradient decreased significantly from a preoperative value of 50 ± 17 mmHg to an intraoperative gradient of 9 ± 4 mmHg (Table 3). The mean gradients were 14, 11, 11, 8 and 6 mmHg for the 19, 21, 23, 25 and 27 mm valve size, respectively. No prosthesis dislocation, endocarditis, valve thrombosis or relevant aortic regurgitation was observed at discharge. CONCLUSIONS The initial experience with the Trifecta valve bioprosthesis shows excellent outcomes with favourable early haemodynamics. Further studies with longer follow-up are needed to confirm those preliminary results.

Initial Clinical Experience With the HeartWare Left Ventricular Assist System: A Single-Center Report

BACKGROUND The HeartWare ventricular assist device (HVAD) system (HeartWare International Inc, Framingham, MA) is a new centrifugal continuous-flow ventricular assist device. The aim of the present study is to review our institutional experience with this novel device. METHODS We reviewed the files of 50 patients (39 men, 11 women) with a mean age of 50.6 ± 11.8 years (range, 19 to 70 years) who underwent HVAD implantation between July 2009 and November 2011. Two patients underwent HeartWare BIVAD implantation. The underlying heart diseases were end-stage ischemic heart disease (n = 12), acute myocardial infarction (n = 9), dilated cardiomyopathy (n = 27) and acute myocarditis (n = 2). Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS) profiles were level 1 (n = 11), 2 (n = 5), 3 (n = 10), and 4 (n = 24). RESULTS After a cumulative support duration of 11,086 days, Kaplan-Meier analysis revealed a survival of 82.0%, 77.9%, 75.5%, at 1, 12, and 24 months, respectively. Causes of early death were right heart failure (n = 4), multiorgan failure (n = 2), septic shock (n = 2), and major neurologic complications (n = 4). One late death occurred due to a right heart failure. Comparison between patients operated on in cardiogenic shock (INTERMACS 1 and 2) and patients who underwent elective HVAD implantation (INTERMACS 3 and 4) revealed a survival of 61.5% and 44.1% for the INTERMACS 1 and 2 group and 90.3% and 87.1% for the INTERMACS 3 and 4 group at 1 and 12 months, respectively (odds ratio, 4.67; p = 0.003). One patient was weaned from the system after 2 years. Eleven patients (22%) were successfully bridged to transplantation. Mean time to transplantation was 209 days (range, 72 to 427 days). Posttransplant survival at the 1-year follow-up was 90.9% (11 patients). CONCLUSIONS Our experience with HVAD shows satisfying results with an excellent posttransplantation survival. Moreover, the stratified survival based on the level of preoperative stability shows better outcomes in patients undergoing elective HVAD implantation.

Contributory Role of Fluorine 18-Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography in the Diagnosis and Clinical Management of Infections in Patients Supported With a Continuous-Flow Left Ventricular Assist Device

BACKGROUND The current study sought to demonstrate the advantages offered by fluorine 18-fluorodeoxyglucose ((18)F-FDG) positron emission tomography/computed tomography (PET/CT) in patients supported with continuous-flow left ventricular assist devices (CF-LVADs) in detecting infection and the consequent effect on clinical decisions. METHODS Between April 2009 and September 2013, 40 PET examinations were performed in 31 patients (78.1% men; mean age, 51.0 ± 14.9 years) supported with a CF-LVAD. In group A (19 examinations in 14 patients), PET/CT was performed to detect infectious focus in patients without external signs of driveline involvement but with at least two of the following infection signs: recurrent fever, positive blood culture, or elevated infectious indicators. In group B (21 examinations in 17 patients), PET/CT aimed to assess the internal extension of infection in patients with external signs of driveline infection. RESULTS In 50% of the cases of the patients in group A, abnormal (18)F-FDG uptake (9 patients) was related to VAD components. Matching the results with the final diagnosis, we reported 9 true-positive, 8 true-negative, no false-negative, and 2 false-positive findings. New information unrelated to VAD was found in 9 patients (50%): pneumonia in 3, colon diverticulitis in 3, sternal dehiscence in 1, paravertebral abscess in 1, and erysipelas in 1. In group B, superficial abnormal (18)F-FDG uptake was found at the piercing site of the driveline in 2 patients, deeper extension of infection along the driveline in 10, initial involvement of the pump housing in 2, and full involvement of the device in 4. These findings contributed to changing the clinical management in 84.2% of group A patients and in 85.7% of group B patients: 16 patients were scheduled for urgent transplantation, 2 underwent surgical revision of the driveline, 7 required prolonged antibiotic therapy, and 3 underwent colonoscopy. CONCLUSIONS This single-center experience highlights the diagnostic value of PET/CT in detecting the localization and internal extension of infection to internal VAD components. Moreover, this information notably influences the therapeutic management.

Direct evidence for the importance of p130 in injury response and arterial remodeling following carotid artery ligation

OBJECTIVE Remodeling of arterial morphology in atherosclerosis, hypertension, and restenosis following angioplasty involves controlled alterations in total vascular circumference which critically modulate sequelae of changes in vessel wall mass. Despite the clinical relevance of this process little is known about the pathophysiology, especially the correlation between smooth muscle cell proliferation and remodeling. METHODS Carotid artery ligation was applied to mice with targeted disruption of the p130 gene (p130 -/-). Mice were allowed to recover for 3 weeks after ligation and then perfusion fixed for histologic and morphometric analysis. RESULTS P130 -/- mice were indistinguishable from control littermates concerning size and weight. As for the aorta, carotid arteries and femoral arteries, no significant differences were found between the groups with regard to vessel size and cellular density of the vessel wall of non-instrumented vessels. In contrast, following carotid artery ligation we found p130 -/- mice (n=8) to develop a significant increase in vessel wall area compared to controls (n=9). Mean values ranged from 3.07 x 10(-2)+/-0.20 x 10(-2)-3.56 x 10(-2)+/-0.62 x 10(-2) mm(2) for p130 -/- mice versus 2.26 x 10(-2)+/-0.13 x 10(-2)-2.57 x 10(-2)+/-0.26 x 10(-2) mm(2) for controls (p=0.02) along the lesion studied. This increase in vessel wall area was primarily due to a sevenfold mean increase in neointima in p130 -/- mice yielding mean values of 0.43+/-0.18 - 1.19+/-0.70 x 10(-2) mm(2). Remarkably, despite vessel wall increase, the lumen area was not statistically different for both groups. CONCLUSIONS The data indicate that the loss of the cell cycle inhibitor p130 leads to an enhanced injury response, implicating a central role of p130 in cell cycle control during response to injury in the vessel wall. The enhanced injury response in the context of p130 -/- preserves the ability to perform perfect remodeling, thus the remodeling capacity is preserved even in the context of this injury model.

Upregulation of connexin43 gap junctions between neointimal smooth muscle cells

Increased expression of connexin43 gap junctions in smooth muscle cells (SMC) is implicated in the response to primary arterial injury and in the early stages of human coronary atherosclerosis, but the relevance of these findings to restenosis is unknown. Here we investigated the expression of connexin43 gap junctions in restenotic aortas of cholesterol-fed double injured rabbits. Immunofluorescence confocal microscopy was used to evaluate temporal and spatial expression patterns and to characterize the major expressing cell type. Parallel studies were conducted by electron microscopy, in situ hybridization and Northern blot analysis. Connexin43 gap junctions- and connexin43 mRNA-expressing cells were abundant in the media of non-injured control aorta. Following primary injury and 6 weeks cholesterol diet, connexin43 gap junctions were found distributed throughout the primary intimal layer; although medial expression was reduced, the overall mRNA expression level remained similar to that of non-injured controls. After secondary injury, no major change in distribution pattern of connexin43 gap junctions occurred up to day 10, when marked neointimal labeling was observed. This overall pattern persisted, though with some diminution, at later stages. On the mRNA level total connexin43 mRNA expression declined to about 40% of control values within 4 days after secondary injury (P < 0.05), but subsequently increased four-fold, attaining levels double that of non-injured controls in the 10-day group (P < 0.005 versus control and 4 days). At later stages mRNA expression levels returned to values similar to those of non-injured controls. At all stages, connexin43 gap junctions were localized to the SMC, not to macrophages. We conclude that the enhanced gap junction formation may contribute to the coordination of the response of SMC after secondary injury, particularly in the early phase of restenosis.

A Recurrent Activating PLCG1 Mutation in Cardiac Angiosarcomas Increases Apoptosis Resistance and Invasiveness of Endothelial Cells

Primary cardiac angiosarcomas are rare tumors with unfavorable prognosis. Pathogenic driver mutations are largely unknown. We therefore analyzed a collection of cases for genomic aberrations using SNP arrays and targeted next-generation sequencing (tNGS) of oncogenes and tumor-suppressor genes. Recurrent gains of chromosome 1q and a small region of chromosome 4 encompassing KDR and KIT were identified by SNP array analysis. Repeatedly mutated genes identified by tNGS were KDR with different nonsynonymous mutations, MLL2 with different nonsense mutations, and PLCG1 with a recurrent nonsynonymous mutation (R707Q) in the highly conserved autoinhibitory SH2 domain in three of 10 cases. PLCγ1 is usually activated by Y783 phosphorylation and activates protein kinase C and Ca(2+)-dependent second messengers, with effects on cellular proliferation, migration, and invasiveness. Ectopic expression of the PLCγ1-R707Q mutant in endothelial cells revealed reduced PLCγ1-Y783 phosphorylation with concomitant increased c-RAF/MEK/ERK1/2 phosphorylation, increased IP3 amounts, and increased Ca(2+)-dependent calcineurin activation compared with ectopic expressed PLCγ1-wild-type. Furthermore, cofilin, whose activation is associated with actin skeleton reorganization, showed decreased phosphorylation, and thus activation after expression of PLCγ1-R707Q compared with PLCγ1-wild-type. At the cellular level, expression of PLCγ1-R707Q in endothelial cells had no influence on proliferation rate, but increased apoptosis resistance and migration and invasiveness in in vitro assays. Together, these findings indicate that the PLCγ1-R707Q mutation causes constitutive activation of PLCγ1 and may represent an alternative way of activation of KDR/PLCγ1 signaling besides KDR activation in angiosarcomas, with implications for VEGF/KDR targeted therapies.

Alterations in the vascular extracellular matrix of granulocyte macrophage colony-stimulating factor (GM-CSF) -deficient mice

GM‐CSF takes part in the cytokine network regulating the metabolism of extracellular matrix (ECM) during atherogenesis. Since data also point to an effect of GM‐CSF on the vascular ECM in general, the vascular collagenous matrix was studied in wild‐type and GM‐CSF‐deficient mice. Histological examination revealed a disorganized vascular ECM in GM‐CSF‐deficient mice involving the collagenous matrix and elastic fiber system. As shown by electron microscopy, collagen bundles were disrupted and reduced. The diameter of fibrils varied widely. mRNA expression of collagens and related molecules was studied. Fibrillar collagens were markedly reduced, α1(I)procollagen to 16.5% of control levels α1(III)procollagen was abolished whereas the expression level of network‐forming α1(VIII)procollagen was not altered. As shown by in situ hybridization, the number of collagen‐expressing cells was reduced. Matrix metalloproteinases and their inhibitor 1 were not affected by GM‐CSF deficiency. Our studies demonstrate that GM‐CSF plays a major role in the cytokine network regulating the metabolism of vascular collagens. GM‐CSF deficiency leads to an altered composition of the vascular collagenous matrix, i.e., reduced amount of fibrillar collagen, altered ratio of fibrillar and network‐forming collagen, and failures in the fibrillogenesis. We suggest that GM‐CSF is a basic requirement for the maintenance of vessel wall integrity and resilience.—Plenz, G., Eschert, H., Beissert, S., Arps, V., Sindermann, J. R., Robenek, H., Völker, W. Alterations in the vascular extracellular matrix of granulocyte macrophage colony‐stimulating factor (GM‐CSF) ‐deficient mice, FASEB J. 17, 1451–1457 (2003)

Paclitaxel and cyclosporine A show supra-additive antiproliferative effects on smooth muscle cells by activation of protein kinase C

Abstract We intended to establish a pharmacologic concept of synergistic antiproliferative effects on smooth muscle cells (SMC) by using paclitaxel and cyclosporine A at clinically applicable doses. Coronary SMC were incubated with paclitaxel and cyclosporine A at concentrations of 10 – 20 nmol/L and 83 – 415 nmol/L, respectively. Antiproliferative effects were assessed by cell counts, [3H]thymidine incorporation and cell cycle analysis. In addition, apoptosis was studied by cytoplasmic histone-associated DNA fragments and in vitro protein kinase C activity (PKC) was determined by immunoassay. We found paclitaxel and cyclosporine A to exert a highly supra-additive antiproliferative effect on SMC with significant reductions of cell counts (p < 0.01) and [3H]thymidine incorporation (p < 0.05). SMC were found to be arrested at the G2/M transition. This antiproliferative effect was observed in the absence of DNA fragmentation above values obtained for single compound treatment, which had virtually no impact on cell proliferation. DNA fragmentation started to increase at a drug combination comprising paclitaxel at the higher dose of 20 nmol/L. Under the treatment with both paclitaxel and cyclosporine A, PKC activity showed a 1.8-fold increase (p < 0.05) compared with untreated controls. In conclusion, PKC mediates supra-additive antiproliferative effects of paclitaxel and cyclosporine A on SMC. The data demonstrate a highly efficient pharmacologic concept for the inhibition of SMC proliferation. Further studies are needed to test this concept under in vivo conditions for the prevention of restenosis or transplant vasculopathy by systemic application of cyclosporine A – when already applied for immunosuppressive purposes – and local delivery of paclitaxel.