Iris Zeller

Cadmium Is a Novel and Independent Risk Factor for Early Atherosclerosis Mechanisms and In Vivo Relevance

Objectives—Although cadmium (Cd) is an important and common environmental pollutant and has been linked to cardiovascular diseases, little is known about its effects in initial stages of atherosclerosis. Methods and Results—In the 195 young healthy women of the Atherosclerosis Risk Factors in Female Youngsters (ARFY) study, cadmium (Cd) level was independently associated with early atherosclerotic vessel wall thickening (intima-media thickness exceeding the 90th percentile of the distribution; multivariable OR 1.6[1.1.–2.3], P=0.016). In line, Cd-fed ApoE knockout mice yielded a significantly increased aortic plaque surface compared to controls (9.5 versus 26.0 mm2, P<0.004). In vitro results indicate that physiological doses of Cd increase vascular endothelial permeability up to 6-fold by (1) inhibition of endothelial cell proliferation, and (2) induction of a caspase-independent but Bcl-xL-inhibitable form of cell death more than 72 hours after Cd addition. Both phenomena are preceded by Cd-induced DNA strand breaks and a cellular DNA damage response. Zinc showed a potent protective effect against deleterious effects of Cd both in the in vitro and human studies. Conclusion—Our research suggests Cd has promoting effects on early human and murine atherosclerosis, which were partly offset by high Zn concentrations.

Ursolic acid causes DNA-damage, P53-mediated, mitochondria- and caspase-dependent human endothelial cell apoptosis, and accelerates atherosclerotic plaque formation in vivo

OBJECTIVE The plant derived triterpene ursolic acid (UA) has been intensively studied in the past; mainly as an anti-cancer compound and for its cardiovascular protective properties. Based on the controversy of reports suggesting anti-angiogenic and cytotoxic effects of UA on one side and cardiovascular and endothelial protective effects on the other side, we decided to assess UA effects on primary human endothelial cells in vitro and atherosclerotic plaque formation in vivo. METHODS AND RESULTS Our in vitro analyses clearly show that UA inhibits endothelial proliferation and is a potent inducer of endothelial cell death. UA causes DNA-damage, followed by the activation of a p53-, BAK-, and caspase-dependent cell-death pathway. Oral application of UA in APO E knockout mice potently stimulated atherosclerotic plaque formation in vivo, which was correlated with decreased serum levels of the athero-protective cytokine IL-5. CONCLUSIONS Due the potent endothelial cell death inducing activity of UA, a systemic application of UA in the treatment of cardiovascular diseases seems unfavourable. UA as an anti-angiogenesis, anti-cancer and - locally applied - cardiovascular drug may be helpful. The DNA damaging activity of UA may however constitute a serious problem.

Identification and pharmacological characterization of the anti-inflammatory principal of the leaves of dwarf elder (Sambucus ebulus L.).

Graphical abstract Anti-inflammatory activity of a dwarf elder leaf extract (Sambucus ebulus L.) was assessed by activity guided fractionation using inhibition of TNFα induced expression of VCAM-1 on the surface of HUVECs as monitoring tool. The active principal was identified as ursolic acid (IC50 6.25 μM).

Characteristics of TAV- and BAV-associated thoracic aortic aneurysms—Smooth muscle cell biology, expression profiling, and histological analyses

OBJECTIVE Past studies on the pathogenesis of thoracic aortic aneurysms have, by concentrating on histological and total tissue analyses, revealed several disease-relevant processes. Despite these studies, there is still a significant lack in the understanding of aneurysmal cell biology today. Hence, it was the goal of this study to assess differences between aneurysmal and healthy aortic smooth muscle cells (SMCs) on a broad - screening-like - basis, allowing us to formulate new hypotheses on the role of SMCs in thoracic aneurysm formation. METHODS AND RESULTS After histological characterization of a total of 16 samples from healthy aortas and thoracic aortic aneurysms (TAA) of patients with bicuspid (BAV) and tricuspid (TAV) aortic valves, we isolated aortic SMCs and subjected them to cell biological and gene expression analyses. The data obtained indicate that aneurysmal SMCs exert reduced proliferation and migration rates compared to controls. BAV TAA SMCs have significantly shorter telomeres, whereas TAV TAA SMCs showed a reduced metabolic activity. In BAV TAA SMCs osteopontin (OPN) expression was significantly elevated, and TAV TAA SMCs showed decreased expression of tissue inhibitor of metalloproteinase 3 (TIMP3). CONCLUSION Our study provides evidence that TAA-associated aortic wall disintegration in BAV and TAV TAAs shows similarities, but also significant differences. BAV and TAV TAAs differ with regard to medial elastic fiber mass and the occurrence of fibroblasts, SMC telomere length, metabolism, and gene expression. This study may form the basis for future in-depth analyses on the relevance of these findings in the pathophysiology of BAV and TAV TAAs.

Leoligin, the major lignan from Edelweiss, inhibits intimal hyperplasia of venous bypass grafts

Aims Despite the lower patency of venous compared with arterial coronary artery bypass grafts, ∼50% of grafts used are saphenous vein conduits because of their easier accessibility. In a search for ways to increase venous graft patency, we applied the results of a previous pharmacological study screening for non-toxic compounds that inhibit intimal hyperplasia of saphenous vein conduits in organ cultures. Here we analyse the effects and mechanism of action of leoligin [(2S,3R,4R)-4-(3,4-dimethoxybenzyl)-2-(3,4-dimethoxyphenyl)tetrahydrofuran-3-yl]methyl (2Z)-2-methylbut-2-enoat, the major lignan from Edelweiss (Leontopodium alpinum Cass.). Methods and results We found that leoligin potently inhibits vascular smooth muscle cell (SMC) proliferation by inducing cell cycle arrest in the G1-phase. Leoligin induced cell death neither in SMCs nor, more importantly, in endothelial cells. In a human saphenous vein organ culture model for graft disease, leoligin potently inhibited intimal hyperplasia, and even reversed graft disease in pre-damaged vessels. Furthermore, in an in vivo mouse model for venous bypass graft disease, leoligin potently inhibited intimal hyperplasia. Conclusion Our data suggest that leoligin might represent a novel non-toxic, non-thrombogenic, endothelial integrity preserving candidate drug for the treatment of vein graft disease.

Lead Contributes to Arterial Intimal Hyperplasia Through Nuclear Factor Erythroid 2–Related Factor–Mediated Endothelial Interleukin 8 Synthesis and Subsequent Invasion of Smooth Muscle Cells

Objective—To validate the hypothesis that the toxic heavy metal lead (Pb) may be linked to cardiovascular diseases via the initiation of atherosclerosis, in vivo and in vitro studies were conducted. Methods and Results—During the human study part of this project, serum Pb levels of healthy young women were correlated to carotid intima-media thickness. Multivariate logistic regression analyses showed that increased serum Pb levels were significantly associated with an increased intima-media thickness (P=0.01; odds ratio per SD unit, 1.6 [95% CI, 1.1 to 2.4]). In vitro, Pb induced an increase in interleukin 8 production and secretion by vascular endothelial cells. Nuclear factor erythroid 2–related factor–2 is the crucial transcription factor involved in Pb-induced upregulation of interleukin 8. Endothelial cell–secreted interleukin 8 triggered intimal invasion of smooth muscle cells and enhanced intimal thickening in an arterial organ culture model. This phenomenon was further enhanced by Pb-increased elastin synthesis of smooth muscle cells. Conclusion—Our data support the hypothesis that Pb is a novel, independent, and significant risk factor for intimal hyperplasia.

Inhibition of cell surface expression of endothelial adhesion molecules by ursolic acid prevents intimal hyperplasia of venous bypass grafts in rats

OBJECTIVES Despite rapid progress in surgical techniques, there is still a significant lack of surgery-supportive pharmacological treatments. The aim of this study was to test the hypothesis that ursolic acid (UA) may prevent intimal hyperplasia of venous bypass grafts. METHODS The hypothesis was tested by means of primary cell isolation and culture followed by real-time polymerase chain reaction, western blotting, fluorescence microscopy and fluorescence-activated cell sorting analyses, as well as an in vivo rat model for intimal hyperplasia of venous bypass grafts and immunohistochemistry and histochemistry. RESULTS The local application of UA significantly inhibited intimal hyperplasia in vivo (intimal thickness control: 25 µm, UA group: 18 µM-8 weeks after surgery). The UA treatment of grafts significantly resulted in reduced endothelial vascular cell adhesion molecule-1 (VCAM-1) expression, reduced infiltration of the grafts vessel wall by CD45-positive cells and increased smooth muscle cell (SMC) death. In in vitro condition, it could be shown that UA inhibits VCAM-1 expression downstream of NFκB and is likely to interfere with VCAM-1 protein synthesis in endothelial cells. Quantification of cell death in vascular smooth muscle cells treated with UA indicated that UA is a potent inducer of SMC apoptosis. CONCLUSIONS Our results suggest that UA-mediated inhibition of endothelial VCAM-1 expression reduces the infiltration of venous bypass grafts by CD45-positive cells and inhibits intimal hyperplasia. Apoptosis induction in SMCs may be another method in which UA reduces intimal thickening. UA may constitute a surgery-supportive pharmacon that reduces intimal hyperplasia of vein grafts.

A Retrospective Assessment of Four Antigen Assays for the Detection of Invasive Candidiasis Among High-Risk Hospitalized Patients

Because of their high mortality rates and non-specific symptoms, invasive Candida infections pose a huge diagnostic and therapeutic challenge. In this study, we evaluated the three mannan antigen assays Platelia, Platelia Plus and Serion, and the (1-3)-β-d-glucan assay Fungitell in a group of high-risk (hematological and surgical) patients. Test results of 305 patients hospitalized at the Vienna General Hospital and the University Hospital of Innsbruck were retrospectively analyzed. We assessed the test accuracy by means of descriptive statistics. Nine (2.95%) patients were affected by invasive candidiasis (IC), and 25 (8.2%) patients had a probable/possible infection. The majority of patients (271; 88.9%) showed no signs of infection. The Platelia and Serion mannan assays had a low sensitivity (65% and 52%, respectively), but high specificity (98% for both tests). The newer version of the Platelia assay, the Platelia Plus, had a higher sensitivity (85%) but a lower specificity (89%). The sensitivity of the Fungitell assay was high (100%), while its specificity was low (58%). The positive predictive values were 0.48 for the Platelia and 0.41 for the Serion assay, 0.26 for the Platelia Plus and 0.09 for the Fungitell assay. Our limited, retrospective study suggests the efficacy of mannan assays as screening (Platelia Plus) and confirmatory (Serion) tests, while the Fungitell assay can be used to exclude invasive Candida infections.

Targeted gene expression analyses and immunohistology suggest a pro-proliferative state in tricuspid aortic valve-, and senescence and viral infections in bicuspid aortic valve-associated thoracic aortic aneurysms

BACKGROUND AND AIMS Despite the potential life-threatening consequences of thoracic aortic aneurysms (TAAs), the pathogenesis of these diseases is still poorly understood. While some aspects of TAA formation have been elucidated, the role of vascular smooth muscle cells (SMCs) in both bicuspid aortic valve (BAV)-associated and degenerative tricuspid aortic valve (TAV)-associated TAAs has not yet been fully unravelled. Thus, this work was aimed at uncovering processes in SMC biology that may contribute to TAA formation. METHODS Using isolated SMCs and tissue samples from TAAs linked to BAV syndrome, TAV-associated degenerative TAAs and control aortas, we performed targeted mRNA expression profile analyses and conducted immunohistological analyses on aortic wall tissue sections. RESULTS While SMC expression profiles and tissue analyses in TAV-TAAs clearly point toward a pro-proliferative state of the aortic media SMCs, BAV-TAA SMCs and tissue provide evidence for DNA damage, DNA damage response signalling as well as profound TLR-3 signalling. CONCLUSIONS The data presented in this study emphasizes the importance of SMCs in TAA development. Furthermore, our results provide evidence that the state of SMCs in the BAV-TAA (senescent) and TAV-TAA (pro-proliferative) differs significantly. For the first time, we also present findings that may argue for the occurrence of a viral infection in BAV-TAA SMCs.

Detection of fungal pathogens by a new broad range real-time PCR assay targeting the fungal ITS2 region

Purpose. The rise in the incidence of fungal infections and the expanding spectrum of fungal pathogens make early and broad detection of fungal pathogens essential. In the present study, a panfungal real‐time PCR assay for the broad‐range detection of fungal DNA (Fungi assay) in a wide variety of clinical specimens was developed. Methodology. Our in‐house, HybProbe real‐time PCR assay targets the ITS2 region of fungal DNA. The applicability was evaluated by testing 105 clinical samples from 98 patients with suspected fungal infection. Samples included tissue biopsies, paraffin embedded tissues, aspirates, EDTA‐anticoagulated blood, cerebrospinal fluids and bronchoalveolar lavages. Results. Fungal pathogens were identified by the Fungi assay in 47 samples. In all of these cases, conventional methods and clinical data were also indicative for a fungal infection. Five samples were interpreted false negative. blast analyses of the amplicons derived from 11 samples revealed the presence of environmental fungal species while other tests and clinical data did not suggest a fungal infection. This fact might indicate contaminated samples. The remaining 42 samples were negative by the Fungi assay as well as the conventional methods and were therefore regarded as true negatives. Thus, sensitivity was 90.4% and specificity 79.2 %. Conclusion. The Fungi assay improved the targeted diagnosis of fungal infections allowing pathogen identification in samples that were histologically positive but culture negative. For reliable diagnosis, results have to be interpreted in context with conventional methods and clinical data.