Franziska Schlegel

Allium ursinum L.: Bioassay-Guided Isolation and Identification of a Galactolipid and a Phytosterol Exerting Antiaggregatory Effects

Aims: We wanted to investigate the possible antithrombotic effects and elucidate the chemical identity of the active principles involved in inhibitory effects against adenosine diphosphate (ADP)-induced aggregation of human platelets by wild garlic, Allium ursinum L. Methods: For this purpose, a bioassay-guided isolation procedure was used followed by spectrometric identification of pure active compounds. For the bioassay, blood was taken from healthy human volunteers and platelet-rich plasma was prepared for turbidimetric platelet aggregation tests. Platelet-rich plasma, stimulated with 20 µmol/l of ADP, was treated with extracts of different polarities, fractions and isolated single compounds from A. ursinum. The extracts were investigated by thin-layer chromatography (TLC), HPLC, mass spectroscopy, electrospray ionization mass spectrometry (ESI-MS) and 1/2-dimensional 1H/13C-nuclear magnetic resonance (NMR) spectroscopic techniques. Results: Fresh A. ursinum leaves were extracted with ethanol, which was the potent form that effectively inhibited ADP-induced aggregation of human platelets. This ethanolic extract was subjected to liquid-liquid partition. Whilst the aqueous phase, containing the moiety of cysteine sulphoxide and thiosulphinate derivatives, showed only weak activity on platelet aggregation, the ethyl-acetate and particularly the chloroform partitions showed the highest aggregation-inhibiting potency. Thus, in our bioassay, the effects of alliins/allicins could be neglected. The chloroform phase, possessing the strongest activity, was separated into 28 fractions by gradient-elution open column chromatography on silica gel. The most active fractions 11–17 were separated again, yielding 10 subfractions. This afforded 1,2-di-O-α-linolenoyl-3-O-β-D-galactopyranosyl-sn-glycerol and β-sitosterol-3-O-β-D-glucopyranoside, the structures of which were determined by ESI-MS and 1/2-dimensional 1H/13C NMR spectroscopic techniques. Furthermore, the minute amounts of volatile oil of A. ursinum leaves obtained by steam distillation according to Ph. Eur. could be evaluated as a third aggregation-inhibiting principle. Conclusion: In our study, for the first time, 2 active, non-sulphur-containing constituents of wild garlic, namely a galactolipid and a phytosterol, could be identified exhibiting inhibitory action on ADP-induced aggregation in human blood platelets. As a major constituent, the galactolipid, 1,2-di-O-α-linolenoyl-3-O-β-D-galactopyranosyl-sn-glycerol, not yet found in Allium sp., appears as a new, highly useful marker substance for A. ursinum drugs, or their pharmaceutical or food preparations, as shown by our orientating TLC analyses.

Inverse Relationship between Tumor Proliferation Markers and Connexin Expression in a Malignant Cardiac Tumor Originating from Mesenchymal Stem Cell Engineered Tissue in a Rat in vivo Model

Background: Recently, we demonstrated the beneficial effects of engineered heart tissues for the treatment of dilated cardiomyopathy in rats. For further development of this technique we started to produce engineered tissue (ET) from mesenchymal stem cells. Interestingly, we observed a malignant tumor invading the heart with an inverse relationship between proliferation markers and connexin expression. Methods: Commercial CD54+/CD90+/CD34−/CD45− bone marrow derived mesenchymal rat stem cells (cBM-MSC), characterized were used for production of mesenchymal stem-cell-ET (MSC-ET) by suspending them in a collagen I, matrigel-mixture and cultivating for 14 days with electrical stimulation. Three MSC-ET were implanted around the beating heart of adult rats for days. Another three MSC-ET were produced from freshly isolated rat bone marrow derived stem cells (sBM-MSC). Results: Three weeks after implantation of the MSC-ETs the hearts were surgically excised. While in 5/6 cases the ET was clearly distinguishable and was found as a ring containing mostly connective tissue around the heart, in 1/6 the heart was completely surrounded by a huge, undifferentiated, pleomorphic tumor originating from the cMSC-ET (cBM-MSC), classified as a high grade malignant sarcoma. Quantitatively we found a clear inverse relationship between cardiac connexin expression (Cx43, Cx40, or Cx45) and increased Ki-67 expression (Cx43: p < 0.0001, Cx45: p < 0.03, Cx40: p < 0.014). At the tumor-heart border there were significantly more Ki-67 positive cells (p = 0.001), and only 2% Cx45 and Ki-67-expressing cells, while the other connexins were nearly completely absent (p < 0.0001). Conclusion and Hypothesis: These observations strongly suggest the hypothesis, that invasive tumor growth is accompanied by reduction in connexins. This implicates that gap junction communication between tumor and normal tissue is reduced or absent, which could mean that growth and differentiation signals can not be exchanged.

Injectable tissue engineered pulmonary heart valve implantation into the pig model: A feasibility study.

Background Transcatheter pulmonary valve replacement is currently performed in clinical trials, but is limited by the use of glutaraldehyde-treated bioprostheses. This feasibility study was performed to evaluate delivery-related tissue distortion during implantation of tissue-engineered (TE) heart valves. Material/Methods The injectable TE heart valve was mounted on a self-expanding nitinol stent (n=7) and delivered into the pulmonary position in 7 pigs, (weight 26 to 31 kg), performing a sternotomy or limited lateral thoracotomy. Prior to implantation, the injectable TE heart valves were crimped and inserted into an applicator. Positioning of the implants was guided by fluoroscopy, and after careful deployment, angiographic examination was performed to evaluate the correct delivered position. Hemodynamic measurements were performed by epicardial echocardiography. Finally, the animals were sacrificed and the injectable TE heart valves were inspected by gross examination and histological examination. Results Orthotopic deliveries of the injectable TE heart valves were all successful performed, expect in 1 where the valve migrated due to a discrepancy between pulmonary valve annulus size and injectable TE valve size. Angiographic evaluation (n=6) showed normal valve function, supported by epicardial echocardiography in which no increased flow velocity was measured, neither trans- nor paravalvular regurgitation. Histological evaluation demonstrated absence of tissue damage from the delivery process. Conclusions Transcatheter implantation of an injectable TE heart valve seems to be possible without tissue distortion due to the delivery system.

Effect of Angiotensin(1-7) on Heart Function in an Experimental Rat Model of Obesity

Aim: Obesity is a risk factor for the development of cardiovascular diseases. Recently it was shown that overexpression of the Mas-receptor antagonist angiotensin(1-7) could prevent from diet-induced obesity. However, it remained unclear whether diet-induced obesity and angiotensin(1-7) overexpression might also have effects on the cardiovascular system in these rats. Methods:Twenty three male Sprague Dawley rats were fed with standard chow (SD+chow, n = 5) or a cafeteria diet (SD+CD, n = 6) for 5 months. To investigate the effect of angiotensin(1-7) transgenic rats, expressing an angiotensin(1-7)-producing fusion protein in testis were used. These transgenic rats also received a 5 months feeding period with either chow (TGR+chow, n = 6) or cafeteria diet (TGR+CD, n = 6), respectively. Hemodynamic measurements (pressure-volume loops) were carried out to assess cardiac function and blood pressure. Subsequently, hearts were explanted and investigated according to the Langendorff technique. Furthermore, cardiac remodeling in these animals was investigated histologically. Results:After 5 months cafeteria diet feeding rats showed a significantly increased body weight, which could be prevented in transgenic rats. However, there was no effect on cardiac performance after cafeteria diet in non-transgenic and transgenic rats. Moreover, overexpression of angiotensin(1-7) deteriorated cardiac contractility as indicated by impaired dp/dt. Furthermore, histological analysis revealed that cafeteria diet led to myocardial fibrosis in both, control and transgenic rats and this was not inhibited by an overproduction of angiotensin(1-7). Conclusion:These results indicate that an overexpression of circulating angiotensin(1-7) prevents a cafeteria diet-induced increase in body weight, but does not affect cardiac performance in this experimental rat model of obesity. Furthermore, overexpression of angiotensin(1-7) alone resulted in an impairment of cardiac function.

Reprogramming Bone Marrow Stem Cells to Functional Endothelial Cells in a Mini Pig Animal Model

Background The aims of this study were to compare the morphological, biochemical, and functional properties of reprogrammed bone marrow stem cell (BMSC)-derived arterial endothelial cells (AECs) and venous endothelial cells (VECs), following adenosine triphosphate (ATP)-stimulation in a mini pig animal model. Material/Methods Bone marrow aspiration was performed in six adult mini pigs. Harvested mononuclear cells were isolated, cultured, and treated with vascular endothelial growth factor (VEGF) (16 μg/ml). Transformed cells were characterized using immunofluorescence staining for CD31 and von Willebrandt factor (vWF) and expression of endothelial nitric oxide synthase (eNOS). Cell release of nitric oxide (cNO) was measured using spectrophotometry. Matrigel assays were used to investigate angiogenesis in transformed BMSCs. Results Reprogrammed BMSCs in culture showed a typical cobblestone-like pattern of growth. Immunofluorescence staining was positive for CD31 and vWF expression. Expression of eNOS, using immunofluorescence staining and Western blot, showed no difference between the reprogrammed BMSCs and VECs. Spectrophotometric examination following stimulation with 10mmol/l ATP, showed comparable cNO release for reprogrammed BMSCs (10.87±1.76 pmol/106 cells/min) and VECs (13.23±2.16 pmol/106 cells/min), but reduced cNO release for AECS (3.44±0.75 pmol/106 cells/min). Matrigel assay for angiogenesis showed vascular tube formation of differentiated BMSC endothelial cells (grade 3.25). BMSCs cultured without VEGF did not demonstrate vascular tube formation. Conclusions The findings of this study showed that eNOS expression and release of NO could be used to show that BMSCs can be reprogrammed to functional VECs and AECs.

Evaluation of conventional and frozen elephant trunk techniques on spinal cord blood flow in an animal model

OBJECTIVES The treatment of patients with extensive thoracic aortic disease involving the arch and descending aorta is often performed using the frozen elephant trunk technique (FET). Spinal cord blood flow (SCBF) in cervical, thoracic and lumbar sections prior, during and after aortic arch surgery were compared in conventional elephant trunk (cET) and FET technique in a pig model. METHODS German Landrace pigs (75‐85 kg) underwent aortic arch surgery using the FET (n = 8) or cET (n = 8) techniques. The E‐vita Open hybrid stent graft was applied in all FET animals. Regional SCBF was measured 4 times: (i) before cardiopulmonary bypass, (ii) after 1 h, (iii) after 3 h, and (iv) after 6 h of reperfusion using fluorescence microspheres. Spinal cord segments were examined histopathologically and by immunohistochemistry. RESULTS SCBF in FET decreased significantly from 0.13 ± 0.03 to 0.05 ± 0.02 ml/min/g after 1 h (P = 0.047). While at 3 h of reperfusion, SCBF increased and was comparable to baseline (0.09 ± 0.01 ml/min/g), beyond this time SCBF decreased again (0.05 ± 0.02 ml/min/g). A similar trend was found for SCBF in the cET group (baseline: 0.16 ± 0.04 ml/min/g, 1 h reperfusion: 0.02 ± 0.01 ml/min/g, 3 h reperfusion: 0.03 ± 0.01 ml/min/g and 6 h reperfusion: 0.02 ± 0.01 ml/min/g, P = 0.019). Cervical, thoracic and lumbar SCBF were also comparable in both groups. Histological analyses of spinal cord showed no differences in necrosis between cET and FET, while no differences were found for hypoxia‐inducible factor‐1&agr; and apoptosis‐inducing factor. In contrast, oxidative stress and caspase‐induced apoptosis were higher in cET versus FET. CONCLUSIONS The SCBF changed significantly during extensive aortic arch surgery with circulatory arrest and moderate hypothermia, but such changes were comparable between the FET and cET groups. The implantation of hybrid stent graft did not influence SCBF in thoracic and lumbar segments of the spinal cord. The immunohistological examination showed no differences between cET and FET regarding ischaemic damage and hypoxia‐induced effects in spinal cord segments.

Early Effects in Perivascular Nerves and Arterial Media Following Renal Artery Denervation

In the past years 2 different innovative methods of hypertension treatments were investigated. The first is the promising examination of the electrical activation of the carotid baroreceptors, which is now in the phase III studies,1,2 and second the usage of selective renal sympathetic denervation (RSD) as an alternative treatment in therapy-resistant hypertension. RSD is thought to be based on alteration of the sympathetic innervation of kidney and secondary effects on the renin–angiotensin–aldosterone system. The renal nerves are located in close vicinity to the renal artery wall. Their activation could be important for the progression of therapy-resistant hypertension. In this context, Krum et al3 showed the proof of principle of a percutaneous, catheter-based trail to destroy renal sympathetic nerves by the introduction of a RF catheter into the lumen of the main renal artery and its subsequent connection to a radiofrequency generator. Until recently, the renal denervation seemed to be a promising therapy option for antihypertensive treatment. However, the simplicity HTN-3 study as recently announced4 indicated a lack of effect of RSD. Nevertheless, at present, the underlying molecular mechanisms remain unclear during RSD. RF injury might damage the tissue by thermal coagulation but may also cause apoptosis as observed in colon cancer.5 Thus, we made up the hypothesis that RSD may induce apoptosis in the perivascular nerves. The initiation of apoptosis is still not entirely understood. One important pathway is the release of apoptosis-inducing factor (AIF) from the mitochondrial intermembrane space together with cytochrome c .6 Subsequently, AIF translocates to the nucleus and induces chromatin decondensation and DNA degradation, likely by endonucleases. Typically, when inducing apoptosis, AIF and caspases act together. However, AIF may induce cell death in a caspase-independent manner.7 The extrinsic pathway …