Tumor Treating Fields Suppression of Ciliogenesis Enhances Temozolomide Toxicity

Tumor Treating Fields (TTFields) are low intensity, alternating intermediate frequency (200kHz) electrical fields that extend survival of glioblastoma patients receiving maintenance temozolomide (TMZ) chemotherapy. How TTFields exert efficacy on cancer over normal cells, or interact with TMZ is unclear. Primary cilia are microtubule-based organelles triggered by extracellular ligands, mechanical and electrical field stimulation, and are capable of promoting cancer growth and TMZ chemoresistance. We found in both low and high grade patient glioma cell lines that TTFields ablated cilia within 24 hours. Halting TTFields treatment led to recovered frequencies of elongated cilia. Cilia on normal primary astrocytes, neurons, and multiciliated/ependymal cells were less affected by TTFields. The TTFields-mediated loss of glioma cilia was partially rescued by chloroquine pretreatment, suggesting the effect is in part due to autophagy activation. We also observed death of ciliated cells during TTFields by live imaging. Notably, TMZ-induced stimulation of ciliogenesis in both adherent cells and gliomaspheres was blocked by TTFields. Moreover, the inhibitory effects of TTFields and TMZ on tumor cell recurrence correlated with the relative timing of TMZ exposure to TTFields and ARL13B+ cilia. Finally, TTFields disrupted cilia in patient tumors treated ex vivo. Our findings suggest TTFields efficacy may depend on the degree of tumor ciliogenesis and relative timing of TMZ treatment.

Increased CaMKII-dependent pro-arrhythmic activity in a novel mouse model of obstructive sleep apnoea

Background Obstructive sleep apnoea (OSA) is frequently associated with atrial arrhythmias, but detailed mechanisms remain elusive. Most recently, we found an increased CaMKII-dependent pro-arrhythmic activity in patients with sleep apnoea. Since patients suffer from various confounding comorbidities, we have developed a novel mouse model of OSA by tongue enlargement. Purpose We tested if mice with OSA exhibit increased atrial CaMKII-dependent pro-arrhythmic activity. Methods Polytetrafluorethylene (PTFE) was injected into the tongue of 12 wild-type (WT) and 10 CaMKII knock-out (CKO) mice. 9 WT and 9 CKO mice were used as control without PTFE injection. Inspiratory flow limitations and apnoeas were monitored during murine sleep phases by whole-body plethysmography (Buxco). After eight weeks, isolated atrial cardiomyocytes were incubated with the Ca-sensitive dye FURA-2 AM for 15 min. Regular Ca transients were elicited by electrical field stimulation (1 Hz, 20 V for 4 ms) using epifluorescence microscopy. Pro-arrhythmic non-stimulated events were defined as deviations from diastolic Ca baseline between two stimulated Ca transients. Results Sonographic measurements revealed a significant increase in mean tongue diameter from (in mm) 3.7±0.1 to 5.1±0.1 after PTFE injection (n=23, p<0.0001). There was a significant correlation between magnitude of tongue diameter and frequency of apnoeas in OSA mice (p=0.046, r2=0.19, Fig. 1A). Interestingly, we observed a significantly increased frequency of pro-arrhythmic events of (in s–1) 0.06±0.01 in WT OSA mice compared to 0.02±0.01 in WT control mice (p=0.047, Fig. 1B). Similar results were observed at higher stimulation frequencies (2 and 4 Hz). There was a significant correlation of pro-arrhythmic events with inspiratory flow limitations (p=0.03, r2=0.24, Fig. 1C) and with the frequency of apnoeas by strong trend (p=0.06, r2=0.18). In contrast, no increase in atrial pro-arrhythmic events was observed in CKO mice after PTFE injection (for CKO mice after PTFE vs. CKO mice without PTFE, 0.03±0.01 s–1 vs. 0.03±0.01 s–1, p=0.89, Fig. 1B). Accordingly, the correlations between pro-arrhythmic events and both inspiratory flow limitations (p=0.36, r2=0.05, Fig. 1C) and apnoeas (p=0.82, r2=0.004) were completely abolished in CKO mice. Conclusion In a novel mouse model of obstructive sleep apnoea, atrial pro-arrhythmic activity was increased in a CaMKII-dependent fashion, which may have therapeutic implications. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Benedikt Schaner / Mr., this work is supported by a research grant of the German Cardiac Society (DGK); Stefan Wagner / Professor, was funded by DFG grants Figure 1

Biomimetic cell-actuated artificial muscle with nanofibrous bundles

Biohybrid artificial muscle produced by integrating living muscle cells and their scaffolds with free movement in vivo is promising for advanced biomedical applications, including cell-based microrobotic systems and therapeutic drug delivery systems. Herein, we provide a biohybrid artificial muscle constructed by integrating living muscle cells and their scaffolds, inspired by bundled myofilaments in skeletal muscle. First, a bundled biohybrid artificial muscle was fabricated by the integration of skeletal muscle cells and hydrophilic polyurethane (HPU)/carbon nanotube (CNT) nanofibers into a fiber shape similar to that of natural skeletal muscle. The HPU/CNT nanofibers provided a stretchable basic backbone of the 3-dimensional fiber structure, which is similar to actin-myosin scaffolds. The incorporated skeletal muscle fibers contribute to the actuation of biohybrid artificial muscle. In fact, electrical field stimulation reversibly leads to the contraction of biohybrid artificial muscle. Therefore, the current development of cell-actuated artificial muscle provides great potential for energy delivery systems as actuators for implantable medibot movement and drug delivery systems. Moreover, the innervation of the biohybrid artificial muscle with motor neurons is of great interest for human-machine interfaces.

​Effect of Ferula Elaeochytris Root Extract on Smooth Muscle Contraction of Prostate Gland in Rat

Background: Smooth muscle contraction and enlargement of the prostate are important targets for the treatment of lower urinary tract symptoms in patients with benign prostatic hyperplasia. The effect of Ferula elaeochytris (FE) root extract on tissues that play a role on fertility such as the prostate has not been demonstrated yet. The aim of this study is to investigate the effects of FE extract on isolated rat prostate tissue induced by electrical field stimulation (EFS) in vitro. Methods: In this experimental study, 48 male rats were randomly divided into 6 groups (n = 8). Groups were consisting FE (20 µl), FE + Adenosine triphosphate (ATP; 50 μM), FE + prazosin (0.3 μM), FE + Calcium (Ca2+; 3 mM and 6 mM), FE + suramin (100 μM), FE + phenylephrine and FE + carbachol. Result: In our study, FE extract inhibited the neurogenic contractions induced by EFS on isolated rat anterior prostate tissue. The presence of suramin and prazosin were not significantly increase the inhibition caused by the FE extract, while Ca2+ and ATP significantly increased the inhibition by the FE extract. (p less than 0.05). In addition, FE extract significantly inhibited phenylephrine and carbachol contractions (p less than 0.05).

Paradoxical effects of acidosis on the noradrenaline-induced and neurogenic constriction of the rat tail artery at low temperatures

Although vasodilatation evoked by acidosis at normal body temperature is well known, the reports regarding effect of acidosis on the reactivity of the isolated arteries at low temperatures are non-existent. This study tested the hypothesis that the inhibitory effect of acidosis on the neurogenic vasoconstriction may be increased by cooling. Using wire myography, we recorded the neurogenic contraction of the rat tail artery segments to the electrical field stimulation in the absence and in the presence of 0.03-10.0 µmol/L noradrenaline. The experiments were conducted at 37oC or 25oC and pH 7.4 or 6.6 which was decreased by means of CO2. Noradrenaline at concentration of 0.03-0.1 µmol/L significantly potentiated the neurogenic vasoconstriction at 25oC, and the potentiation was not inhibited by acidosis. Contrary to our hypothesis, acidosis at a low temperature did not affect the noradrenaline-induced tone and significantly increased the neurogenic contraction of the artery segments in the absence and presence of noradrenaline. These effects of acidosis were partly dependent on the endothelium and L-type Ca2+ channels activation. The phenomenon described for the first time might be of importance for the reduction in the heat loss by virtue of decrease in the subcutaneous blood flow at low ambient temperatures.

Restoring Perivascular Adipose Tissue Function in Obesity Using Exercise

PurposePerivascular adipose tissue (PVAT) exerts an anti-contractile effect which is vital in regulating vascular tone. This effect is mediated via sympathetic nervous stimulation of PVAT by a mechanism which involves noradrenaline uptake through organic cation transporter 3 (OCT3) and β3-adrenoceptor-mediated adiponectin release. In obesity, autonomic dysfunction occurs, which may result in a loss of PVAT function and subsequent vascular disease. Accordingly, we have investigated abnormalities in obese PVAT, and the potential for exercise in restoring function.MethodsVascular contractility to electrical field stimulation (EFS) was assessed ex vivo in the presence of pharmacological tools in ±PVAT vessels from obese and exercised obese mice. Immunohistochemistry was used to detect changes in expression of β3-adrenoceptors, OCT3 and tumour necrosis factor-α (TNFα) in PVAT.ResultsHigh fat feeding induced hypertension, hyperglycaemia, and hyperinsulinaemia, which was reversed using exercise, independent of weight loss. Obesity induced a loss of the PVAT anti-contractile effect, which could not be restored via β3-adrenoceptor activation. Moreover, adiponectin no longer exerts vasodilation. Additionally, exercise reversed PVAT dysfunction in obesity by reducing inflammation of PVAT and increasing β3-adrenoceptor and OCT3 expression, which were downregulated in obesity. Furthermore, the vasodilator effects of adiponectin were restored.ConclusionLoss of neutrally mediated PVAT anti-contractile function in obesity will contribute to the development of hypertension and type II diabetes. Exercise training will restore function and treat the vascular complications of obesity.

Effects of High Salt Intake on Detrusor Muscle Contraction in Dahl Salt-Sensitive Rats

High salt intake has been reported as a risk factor for urinary storage symptoms. However, the association between high salt intake and detrusor muscle contraction is not clear. Therefore, we investigated the effects of high salt intake on the components of detrusor muscle contraction in rats. Six-week-old male Dahl salt-resistant (DR; n = 5) and Dahl salt-sensitive (DS; n = 5) rats were fed a high salt (8% NaCl) diet for one week. The contractile responses of the detrusor muscle to the cumulative administration of carbachol and electrical field stimulation (EFS) with and without suramin and atropine were evaluated via isometric tension study. The concentration–response curves of carbachol were shifted more to the left in the DS group than those in the DR group. Contractile responses to EFS were more enhanced in the DS group than those in the DR group (p < 0.05). Cholinergic component-induced responses were more enhanced in the DS group than those in the DR group (p < 0.05). High salt intake might cause urinary storage symptoms via abnormalities in detrusor muscle contraction and the enhancement of cholinergic signals. Excessive salt intake should be avoided to preserve bladder function.

Vasomotor action of androgens in the mesenteric artery of hypertensive rats. Role of perivascular innervation

Androgens may exert cardiovascular protective actions by regulating the release and function of different vascular factors. In addition, testosterone (TES) and its 5-reduced metabolites, 5α- and 5β-dihydrotestosterone (5α- and 5β-DHT) induce vasorelaxant and hypotensive effects. Furthermore, hypertension has been reported to alter the release and function of the neurotransmitters nitric oxide (NO), calcitonin gene-related peptide (CGRP) and noradrenaline (NA). Since the mesenteric arteries possess a dense perivascular innervation and significantly regulate total peripheral vascular resistance, the objective of this study was to analyze the effect of TES, 5α- and 5β-DHT on the neurogenic release and vasomotor function of NO, CGRP and NA. For this purpose, the superior mesenteric artery from male spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats was used to analyze: (i) the effect of androgens (10 nM, incubated for 30 min) on the neurogenic release of NO, CGRP and NA and (ii) the vasoconstrictor-response to NA and the vasodilator responses to the NO donor, sodium nitroprusside (SNP) and exogenous CGRP. The results showed that TES, 5α- or 5β-DHT did not modify the release of NO, CGRP or NA induced by electrical field stimulation (EFS) in the arteries of SHR; however, in the arteries of WKY rats androgens only caused an increase in EFS-induced NO release. Moreover, TES, and especially 5β-DHT, increased the vasodilator response induced by SNP and CGRP in the arteries of SHR. These findings could be contributing to the hypotensive/antihypertensive efficacy of 5β-DHT previously described in conscious SHR and WKY rats, pointing to 5β- DHT as a potential drug for the treatment of hypertension.

First person – José Britto-Júnior

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. José Britto-Júnior is first author on ‘The basal release of endothelium-derived catecholamines regulates the contractions of Chelonoidis carbonaria aorta caused by electrical-field stimulation’, published in BiO. José conducted the research described in this article while a master's student in Professor Matheus L. Rocha's laboratory at Faculty of Pharmacy, University of Goias, and is now a PhD student in the Department of Pharmacology at the University of Campinas, Brasil, investigating basic cardiovascular pharmacology, endothelium, endothelial catecholamines and comparative physiology.