Gorazd Drevenšek

The endothelial plasma membrane transporter bilitranslocase mediates rat aortic vasodilation induced by anthocyanins

BACKGROUND AND AIMS Anthocyanins, a sub-class of flavonoids, induce endothelium-dependent vasorelaxation, by activating endothelial nitric oxide synthase and consequently increasing production of the vasorelaxant agent nitric oxide. It is not yet clear if anthocyanin-induced vasorelaxation starts with their interaction with plasma membrane receptors in the extracellular compartment, or with their membrane transport toward intracellular molecular targets. We therefore investigated the possible role of bilitranslocase (TC 2.A.65.1.1), an endothelial plasma membrane carrier that transports flavonoids, in the vasodilation activity induced by anthocyanins. METHODS AND RESULTS Vascular reactivity was assessed in thoracic aortic rings obtained from male Wistar rats. Pre-treatment of aortic rings with anti-sequence bilitranslocase antibodies targeting the carrier, decreased vasodilation induced by cyanidin 3-glucoside and bilberry anthocyanins. CONCLUSION Here we show for the first time that bilitranslocase mediates a critical step in vasodilation induced by anthocyanins. This offers new insights into the molecular mechanism involved in endothelium-dependent vasorelaxation by flavonoids, and the importance of their specific membrane carriers.

Effects of equinatoxin II from Actinia equina (L.) on isolated rat heart: The role of direct cardiotoxic effects in equinatoxin II lethality

Equinatoxin II is a lethal basic protein isolated from the sea anemone Actinia equina (L.) with LD50 in mice 35 microg/kg. The putative cause of death is cardiorespiratory arrest, but the mechanism of cardiotoxicity is poorly understood. It is not clear whether the toxin injected intravenously into an experimental animal reaches the heart in a concentration sufficient to cause direct effects on the heart. Therefore experiments were performed on rats and on isolated rat hearts in order to investigate the possible direct cardiotoxic effects of the toxin. For this reason the hearts were perfused with different concentrations of the toxin and with the effluent from the lungs collected during perfusion of the lungs with equinatoxin II. The results revealed the clear dose-dependent, direct cardiotoxic effects of the toxin and of the effluent from the lungs on Langendorffs heart preparations. The threshold concentration of equinatoxin II causing a drop in the perfusion rate, decreased left ventricular pressure, arrhythmia and increased LDH release, was found to be around 0.1 to 1 nM. With 10 nM equinatoxin II the left ventricular pressure dropped to 14+/-11% of the control, and the coronary flow to 9+/-3%. These effects were followed by arrhythmia and cardiac arrest. The concentration of equinatoxin recovered from the lungs after the perfusion with 100 nM equinatoxin II ranged between 0.8 and 5 nM. The results indicate that direct cardiotoxic effects of equinatoxin II play an important role in the lethal effects of the toxin.

The endothelin system mediates bone modeling in the late stage of orthodontic tooth movement in rats

The endothelin system is involved in orthodontic tooth movement (OTM). The aim of the study was to examine the role of ET-1, ET(A) and ET(B) in bone modeling during OTM in rats. Male Wistar rats (n=62) were divided into three groups: control animals (n=10; control group) without appliance, and two groups of experimental animals, which were applied a super-elastic closed-coil spring between the first left maxillary molar and the incisors and were treated daily with either TBC3214 (n=10; TBC3214 group) or with saline (n=42; appliance only group). TBC3214 is a highly selective antagonist on ET(A) receptors. The distance between teeth was measured on days 0 and 42. On days 0, 14, 28 and 42 animals of the appliance only group (n=8) were sacrificed and tissue samples were taken. Total RNA and protein contents were isolated. Gene expression levels of ET-1, ET(A) and ET(B) were assessed by means of relative RT-PCR. Protein levels of ET(A) and ET(B) were examined by immunoblotting. Ten animals of each group were sacrificed on day 42 and tissue samples were prepared for histological analysis. Alveolar bone volume, osteoblast and osteoclast volume were determined histomorphometrically. Gene expression levels of ET-1, ET(A) and ET(B) varied throughout the experiment and were significantly up-regulated on day 42 (p<0.001). The immunoreactivity of ET(A) and ET(B) significantly decreased on day 14 (p<0.001) and increased on day 28 (p<0.001). Alveolar bone volume was significantly higher in the TBC3214 group compared to the appliance only group (p<0.001). Osteoclast volume was significantly lower in the TBC3214 group compared to the appliance only group (p<0.05). Gene and protein expression levels of ET-1, ET(A) and ET(B) varied significantly during OTM, suggesting their different roles in the various stages of OTM. TBC3214 significantly increased alveolar bone volume and significantly decreased osteoclast volume, indicating that it decreased bone resorption in stage three of OTM. These data suggest that ET-1 increases osteoclastic bone resorption via ET(A) in the late stage of OTM.

Recombinant Single-Chain Antibody with the Trojan Peptide Penetratin Positioned in the Linker Region Enables Cargo Transfer Across the Blood–Brain Barrier

Delivery of therapeutic proteins into tissues and across the blood–brain barrier (BBB) is limited by the size and biochemical properties of the proteins. Efficient delivery across BBB is generally restricted to small, highly lipophilic molecules. However, in the last decades, several peptides that can pass cell membranes have been identified. It has been shown that these peptides are also capable of delivering large hydrophilic cargoes into cells and are therefore a powerful biological tool for transporting drugs across cell membranes and even into the brain. We designed and prepared a single-chain antibody fragment (scFvs), specific for the pathological form of the prion protein (PrPSc), where a cell-penetrating peptide (CPP) was used as a linker between the two variable domains of the scFv. The intravenously administered recombinant scFv-CPP was successfully targeted to and delivered into mouse brain cells. Our single-chain antibody fragments are of special interest in view of possible therapeutic reagents design not only for prion diseases but also for other neurodegenerative diseases.

Low-dose atorvastatin, losartan, and particularly their combination, provide cardiovascular protection in isolated rat heart and aorta

Statins and angiotensin receptor blockers at therapeutic doses have beneficial cardiovascular effects, which can be applied for cardiovascular protection. We explored whether low doses of atorvastatin, losartan, and particularly their combination, possess important pleiotropic vasodilatory effects. Wistar rats were treated daily with low-dose atorvastatin (2 mg/kg, n = 15), low-dose losartan (5 mg/kg, n = 15), their combination (n = 15), or saline (n = 15). After 4, 6, or 8 weeks the animals were anesthetized, blood samples taken, and their hearts and thoracic aortas isolated. Two kinds of experiments were performed: the measurement of coronary flow rate after ischemia/reperfusion myocardial injury and endothelium-dependent relaxation of thoracic aorta. In both models, maximal vasodilation activity was obtained in rats treated for 6 weeks. In the ischemia/reperfusion myocardial injury model, coronary flow increased (atorvastatin or losartan 1.9-fold, P < 0.01; combination 2.4-fold, P < 0.001) compared with controls. In the thoracic aorta model, endothelium-dependent relaxation significantly increased only in the combination group compared with the control group (up to 1.4-fold; P < 0.01). Simultaneously, we detected increased anti-inflammatory activity and increased nitric oxide concentration, but no changes in lipids and blood pressure. In a rat model we showed important vasodilatory activity of low-dose atorvastatin, losartan, and particularly their combination. The effects of the low-dose combination were accompanied by, and probably at least partly achieved by, anti-inflammatory and nitric oxide pathways. Overall, these results could be valuable for the development of new vascular protective strategies focusing on a low-dose regimen of statins and sartans, and particularly their combination.

Treatment With Low-dose Atorvastatin, Losartan and Their Combination Increases Expression of Vasoactive-Related Genes in Rat Aortas

Recently it has been shown that statins and angiotensin receptor blockers (ARBs) at low doses express beneficial pleiotropic vascular effects. We aimed to explore whether these drugs at low doses induce the expression of vasoactive-related genes. Sixty adult Wistar rats were treated with low-dose atorvastatin (2 mg/kg), low-dose losartan (5 mg/kg), their combination or saline daily for 4, 6, or 8 weeks. Expression of the vasoactive-related genes endothelin receptor type A (EDNRA), endothelial nitric oxide synthase 3 (NOS3), inducible nitric oxide synthase 2 (NOS2), and angiotensin II receptor type 1 (AGTRL1a) was measured in isolated thoracic aortas. Expression of EDNRA gradually decreased, the lowest values being obtained after 8 weeks (low-dose atorvastatin, losartan [1.6- and 1-7-fold vs controls, respectively; both P < .05], and the combination [2.3-fold vs control, P < .001]). The highest values of NOS3 were obtained after 6 weeks (low-dose atorvastatin, losartan, and their combination, 3.1-fold, P < .01; 3.4-fold, P < .001; and 3.6-fold, P < .001 vs controls, respectively) and then declined after 8 weeks. The combination was more effective in inducing total NOS3 expression when compared to the separate drugs (1.4-fold; P < .05). Importantly, expression of NOS3 was associated with increased plasma NO levels and positively correlated with thoracic aorta relaxation. No changes in expression of NOS2 and AGTRL1a were observed. We showed that low-dose atorvastatin or losartan and especially their combination increases the expression of NOS3 and decreases the expression of EDNRA. These findings are valuable in explaining the effectiveness of the “low-dose pharmacological approach” for improvement in arterial function.

Expression levels of endothelin-1, endothelin-2, and endothelin-3 vary during the initial, lag, and late phase of orthodontic tooth movement in rats

Endothelins (ET)-1, ET-2, and ET-3 are one group of cytokines likely to be released during orthodontic tooth movement (OTM). Therefore, the expression of ET levels was investigated to determine the importance and involvement of isopeptides during the several phases of OTM. Thirty-two male Wistar rats (12-13 weeks old) were divided into four groups of eight: control, 14, 28, and 42 day groups. Tooth movement was induced by a closed-coil spring inserted between the upper left first molar and the upper incisors. The distance between the teeth was measured on days 0, 2, 7, 14, 21, 28, 35, and 42 using a digital calliper. The rate of tooth movement was calculated. The animals were sacrificed on days 14, 28, and 42 and gene expression levels of all three ET were determined using reverse transcription polymerase chain reaction. Statistical analysis was performed using two-way analysis of variance, Bonferronis correction, and paired t-tests. The distance between the teeth decreased in all appliance groups (P < 0.001). The rate of tooth movement was 0.20 +/- 0.02, 0.03 +/- 0.01, and 0.06 +/- 0.02 mm/day between days 0-2, 3-21, and 22-42, respectively. On day 14, gene expression levels for ET-1 (P < 0.05) and ET-3 (P < 0.001) increased compared with day 0. On day 28, a downregulation of ET-3 was observed when compared with day 0 (P < 0.001). On day 42, ET-1 (P < 0.001) and ET-3 (P < 0.01) gene expression levels were strongly upregulated, while ET-2 gene expression level was downregulated (P < 0.01) when compared with day 0. ET-1 and ET-3, but not ET-2, are involved in all three phases of OTM, and ET-1 seems to be the predominant form in the late phase of OTM.

A low-dose atorvastatin and losartan combination directly improves aortic ring relaxation and diminishes ischaemic-reperfusion injury in isolated rat hearts

Summary Background The cardiovascular pleiotropic effects of statins and angiotensin receptor blockers (ARBs) could be of interest for innovative preventive approaches. We aimed to investigate whether low-dose atorvastatin and losartan, separately not possessing protective cardiovascular pleiotropic effects, express them when combined. Material/Methods Forty-five adult male Wistar rats were anaesthetized and their thoracic aortas and hearts were isolated. Relaxation of aortic rings, coronary flow rate and the extent of myocardial ischaemic-reperfusion injury were measured. Different concentrations (0.01, 0.1, 1.0 μM) of atorvastatin and losartan added to a perfusion medium were first tested. The separate drugs, which were ineffective, were then combined at the same concentrations and the concentration was tested in the same model. Results Low concentrations of atorvastatin or losartan (0.1 and 1 μM, respectively) produced no effects in isolated aorta. However, surprisingly, when these drug concentrations were combined, a significantly improved endothelium-dependent relaxation of the thoracic aorta was observed. Similarly, when combining individually ineffective concentrations of atorvastatin or losartan (0.01 and 0.1 μM, respectively), significantly increased coronary flow and a decreased extent of myocardial injury were observed. By using a nitric oxide-synthase inhibitor, we demonstrated that the vasodilatory effects obtained were nitric oxide-dependent. The degree of effectiveness by the combination was comparable to that obtained by 10-fold (atorvastatin) or 100-fold (losartan) higher concentrations of the separate drugs. Conclusions Our results revealed that remarkable additive/synergistic effects exist between low-doses of a statin (atorvastatin) and an ARB (losartan), resulting in important cardiovascular protection. This new concept could be valuable in cardiovascular prevention.

Orthodontic force decreases the eruption rate of rat incisors.

The aim of this study was to determine whether a force applied in an antero-posterior direction would adequately reduce incisor eruption. This is needed to achieve a constant direction of force which is one of the demands for a good model for studying orthodontic tooth movement. Twenty male Wistar rats aged 11-12 weeks were divided into two equal groups: in the appliance group, a superelastic closed coil spring (25 cN) was placed between the upper left first molar and the incisors. The control group consisted of animals without an appliance. In both groups, cuts were created on the labial surfaces of the upper and lower incisors. The distance from the gingival reference point to the midpoint of the cut was measured for 10 days at 2 day intervals. Upper incisor inclination was determined as the distance from the most mesial point of the upper left first molar to the incisal edge of the ipsilateral incisor on days 0 and 10. Statistical analysis was carried out using two-way analysis of variance and a Bonferroni post- test to estimate reliability. The eruption rates of the maxillary incisors in the appliance group were significantly decreased when compared with the control group during the whole experiment. In the appliance group, the eruption rates of the mandibular incisors were decreased more than those of the maxillary incisors (P<0.01). There was no difference in incisor inclination between the appliance and control groups on day 10 (P=0.81). The applied force of 25 cN in an antero-posterior direction diminished incisor eruption to a level which enabled a constant direction of orthodontic force for 10 days.

Normothermic and hypothermic models for studying the deleterious effects of hypoxia-reoxygenation on EDHF-mediated relaxation in isolated porcine coronary arteries

INTRODUCTION The vasomotor response of the coronary artery is altered by hypoxia-reoxygenation (H-R) induced damage. The aim of our study was to compare and evaluate normothermic and hypothermic models which are suitable for future drug studies of vasoprotective action against H-R injury. METHODS Porcine coronary arterial rings were isolated and placed in Krebs-Henseleit (K-H) solution. Rings were exposed to normoxic conditions (control group) and two different H-R conditions: the first induced by a 95% N(2)-5% CO(2) gas mixture (40- and 60-min hypoxia) in a normothermic protocol, and the second induced by hypothermic (4 degrees C) hypoxia-reoxygenation in an air-tight beaker filled with K-H solution (24- and 48-hours hypoxia). Reoxygenation was applied by introducing K-H solution aerated with a 95% O(2)-5% CO(2) mixture under normothermic (37 degrees C) conditions. To test the EDHF-mediated relaxation by substance P, rings were first incubated in L-NNA, nitric oxide synthase inhibitor, and indomethacin, cyclooxygenase inhibitor, and then pre-contracted with thromboxane analogue U-46619. Analysis of the maximum relaxation of the arterial rings was performed by one-way ANOVA, followed by Bonferronis post-test. RESULTS Distal segments of the coronary artery responded faster to contraction induced by U-46619 and were relaxed by substance P to a greater extent than proximal segments. Maximal relaxations of arterial rings induced by a 10 nM solution of substance P were significantly reduced (p<0.001) from the values for normoxic rings (81.0+/-1.0%, n=30) after 40-min H-R (50.5+/-5.3%, n=30), 60-min H-R (32.1+/-3.5%, n=30), 24-hours hypothermic H-R (56.0+/-2.3%, n=30) and after 48-hours hypothermic H-R (38.5+/-5.1%, n=30). CONCLUSIONS The model employing 40-min normothermic H-R is as effective as 24-hours hypothermic H-R, and 60-min normothermic H-R as 48-hours hypothermic H-R for studying the deleterious effects of H-R on EDHF-mediated relaxation.