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Murine breast cancer feed arteries are thin-walled with reduced ?1A-adrenoceptor expression and attenuated sympathetic vasocontraction.

ABSTRACT: Perfusion of breast cancer tissue limits oxygen availability and metabolism but angiogenesis inhibitors have hitherto been unsuccessful for breast cancer therapy. In order to identify abnormalities and possible therapeutic targets in mature cancer arteries, we here characterize the structure and function of cancer feed arteries and corresponding control arteries from female FVB/N mice with ErbB2-induced breast cancer.We investigated the contractile function of breast cancer feed arteries and matched control arteries by isometric myography and evaluated membrane potentials and intracellular [Ca2+] using sharp electrodes and fluorescence microscopy, respectively. Arterial wall structure is assessed by transmission light microscopy of arteries mounted in wire myographs and by evaluation of histological sections using the unbiased stereological disector technique. We determined the expression of messenger RNA by reverse transcription and quantitative polymerase chain reaction and studied receptor expression by confocal microscopy of arteries labelled with the BODIPY-tagged ?1-adrenoceptor antagonist prazosin.Breast cancer feed arteries are thin-walled and produce lower tension than control arteries of similar diameter in response to norepinephrine, thromboxane-analog U46619, endothelin-1, and depolarization with elevated [K+]. Fewer layers of similarly-sized vascular smooth muscle cells explain the reduced media thickness of breast cancer arteries. Evidenced by lower media stress, norepinephrine-induced and thromboxane-induced tension development of breast cancer arteries is reduced more than is explained by the thinner media. Conversely, media stress during stimulation with endothelin-1 and elevated [K+] is similar between breast cancer and control arteries. Correspondingly, vascular smooth muscle cell depolarizations and intracellular Ca2+ responses are attenuated in breast cancer feed arteries during norepinephrine but not during endothelin-1 stimulation. Protein expression of ?1-adrenoceptors and messenger RNA levels for ?1A-adrenoceptors are lower in breast cancer arteries than control arteries. Sympathetic vasocontraction elicited by electrical field stimulation is inhibited by ?1-adrenoceptor blockade and reduced in breast cancer feed arteries compared to control arteries.Thinner media and lower ?1-adrenoceptor expression weaken contractions of breast cancer feed arteries in response to sympathetic activity. We propose that abnormalities in breast cancer arteries can be exploited to modify tumor perfusion and thereby either starve cancer cells or facilitate drug and oxygen delivery during chemotherapy or radiotherapy.

SUBMITTER: Froelunde AS

PROVIDER: S-EPMC5863844 | biostudies-other | 2018 Mar

REPOSITORIES: biostudies-other

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Murine breast cancer feed arteries are thin-walled with reduced α<sub>1A</sub>-adrenoceptor expression and attenuated sympathetic vasocontraction.

Froelunde Anne Sofie AS Ohlenbusch Marit M Hansen Kristoffer B KB Jessen Nicolai N Kim Sukhan S Boedtkjer Ebbe E

Breast cancer research : BCR 20180322 1

<h4>Background</h4>Perfusion of breast cancer tissue limits oxygen availability and metabolism but angiogenesis inhibitors have hitherto been unsuccessful for breast cancer therapy. In order to identify abnormalities and possible therapeutic targets in mature cancer arteries, we here characterize the structure and function of cancer feed arteries and corresponding control arteries from female FVB/N mice with ErbB2-induced breast cancer.<h4>Methods</h4>We investigated the contractile function of ...[more]

PMID: 29566737

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Project description:Two splice isoforms of rabbit alpha(1a)-adrenergic receptor (AR), (named alpha(1a)-OCU.2-AR and alpha(1a)-OCU.3-AR) have been isolated from the liver cDNA library in addition to the previously reported isoform (alpha(1a)-OCU.1-AR). Although they have the identical splice position with human alpha(1a)-AR isoforms, the C-terminal sequences are distinct from those of human isoforms. Among these rabbit alpha(1a)-AR isoforms, there are no significant differences in pharmacological properties: high affinity for prazosin, WB4101, KMD-3213 and YM617 and low affinity for BMY7378, using COS-7 cells expressing each isoform by radioligand binding assay. Competitive reverse transcription-polymerase chain reaction (RT - PCR) analysis revealed that mRNA of alpha(1a)-ARs was expressed in liver, thoracic aorta, brain stem and thalamus of rabbit. The splice isoforms exhibited a distinct distribution pattern in rabbit; alpha(1a)-OCU. 1-AR was expressed most abundantly in those tissues. CHO clones, stably expressing each isoforms with receptor density 740 fmol mg(-1) protein in alpha(1a)-OCU.1-AR, 1200 fmol mg(-1) in alpha(1a)-OCU.2-AR and 570 fmol mg(-1) in alpha(1a)-OCU.3-AR, respectively, showed a noradrenaline-induced increase in inositol trisphosphate which was suppressed by prazosin. Noradrenaline elicited a concentration-dependent increase in extracellular acidification rate (EAR) in the CHO clones with pEC(50) values of 6. 19 for alpha(1a)-OCU.1-AR, 6.49 for alpha(1a)-OCU.2-AR and 6.58 for alpha(1a)-OCU.3-AR, respectively. Noradrenaline caused a concentration-dependent increase in intracellular Ca(2+) concentration ([Ca(2+)]i) in the CHO clones with pEC(50) values of 6. 14 for alpha(1a)-OCU.1-AR, 7.25 for alpha(1a)-OCU.2-AR and 7.70 for alpha(1a)-OCU.3-AR, respectively. In conclusion, the present study shows the occurrence of three splice isoforms of rabbit alpha(1a)-AR, which are unique in C-terminal sequence and in tissue distribution. They show similar pharmacological profiles in binding studies but alpha(1a)-OCU.3-AR had the highest potency of noradrenaline in functional studies in spite of the lowest receptor density. These findings suggest that the structure of C-terminus of alpha(1a)-ARs may give the characteristic functional profile.

Project description:Background and objectiveSympathetic activity plays an important role in the proliferation and differentiation of stem cells, and it changes over time, thereby exerting differential effects on various stem cell types. Aging causes sympathetic hyperactivity in aged tissues and blunts sympathetic nerves regulation, and sympathetic abnormalities play a role in aging-related diseases. Currently, the effect of sympathetic activity on skeletal muscle stem cells, namely satellite cells (SCs), is unclear. This study aimed to investigate the effects of skeletal muscle sympathetic activity on SC aging and skeletal muscle repair.Materials and methodsTo evaluate skeletal muscle and fibrotic areas, numbers of SCs and myonuclei per muscle fiber, β2-adrenoceptor (β2-ADR) expression, muscle repair, and sympathetic innervation in skeletal muscle, aged mice, young mice that underwent chemical sympathectomy (CS) were utilized. Mice with a tibialis anterior muscle injury were treated by barium chloride (BaCl2) and clenbuterol (CLB) in vivo. SCs or C2C12 cells were differentiated into myotubes and treated with or without CLB. Immunofluorescence, western blot, sirius red, and hematoxylin-eosin were used to evaluate SCs, myogenic repair and differentiation.ResultsThe number of SCs, sympathetic activity, and reparability of muscle injury in aged mice were significantly decreased, compared with those in young mice. The above characteristics of young mice that underwent CS were similar to those of aged mice. While CLB promoted the repair of muscle injury in aged and CS mice and ameliorated the reduction in the SC number and sympathetic activity, the effects of CLB on the SCs and sympathetic nerves in young mice were not significant. CLB inhibited the myogenic differentiation of C2C12 cells in vitro. We further found that NF-κB and ERK1/2 signaling pathways were activated during myogenic differentiation, and this process could be inhibited by CLB.ConclusionNormal sympathetic activity promoted the stemness of SCs to thereby maintain a steady state. It also could maintain total and self-renewing number of SCs and maintain a quiescent state, which was correlated with skeletal SCs via β2-ADR. Normal sympathetic activity was also beneficial for the myogenic repair of injured skeletal muscle.

Project description:BACKGROUND AND PURPOSE: alpha(1)-Adrenoceptors in the rabbit prostate have been studied because of their controversial pharmacological profiles in functional and radioligand binding studies. The purpose of the present study is to determine the native profiles of alpha(1)-adrenoceptor phenotypes and to clarify their relationship. EXPERIMENTAL APPROACH: Binding experiments with [3H]-silodosin and [3H]-prazosin were performed using intact tissue segments and crude membrane preparations of rabbit prostate and the results were compared with alpha(1)-adrenoceptor-mediated prostate contraction. KEY RESULTS: [3H]-Silodosin at subnanomolar concentrations bound specifically to intact tissue segments of rabbit prostate. However, [3H]-prazosin at the same range of concentrations failed to bind to alpha(1)-adrenoceptors of intact segments. Binding sites of [3H]-silodosin in intact segments were composed of alpha(1L) phenotype with low affinities for prazosin (pKi=7.1), 5-methyurapidil and N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha,alpha-dimethyl-1H-indole-3-ethamine hydrochloride (RS-17053), and alpha(1A)-like phenotype with moderate affinity for prazosin (pKi=8.8) but high affinity for 5-methyurapidil and RS-17053. In contrast, both radioligands bound to a single population of alpha(1)-adrenoceptors in the membrane preparations at the same density with a subnanomolar affinity, showing a typical profile of 'classical' alpha(1A)-adrenoceptors (pKi for prazosin=9.8). The pharmacological profile of alpha(1)-adrenoceptor-mediated prostate contraction was in accord with the alpha(1L) phenotype observed by intact segment binding approach. CONCLUSIONS AND IMPLICATIONS: Three distinct phenotypes (alpha(1L) and alpha(1A)-like phenotypes in the intact segments and a classical alpha(1A) phenotype in the membranes) with different affinities for prazosin were detected in rabbit prostate. It appears that the three phenotypes are phenotypic subtypes of alpha(1A)-adrenoceptors, but are not genetically different subtypes.

Project description:Background and purposeIn small arteries, small conductance Ca²?-activated K? channels (SK(Ca)) and intermediate conductance Ca²?-activated K? channels (IK(Ca)) restricted to the vascular endothelium generate hyperpolarization that underpins the NO- and PGI?-independent, endothelium-derived hyperpolarizing factor response that is the predominate endothelial mechanism for vasodilatation. As neuronal IK(Ca) channels can be negatively regulated by PKA, we investigated whether ?-adrenoceptor stimulation, which signals through cAMP/PKA, might influence endothelial cell hyperpolarization and as a result modify the associated vasodilatation.Experimental approachRat isolated small mesenteric arteries were pressurized to measure vasodilatation and endothelial cell [Ca²?]i , mounted in a wire myograph to measure smooth muscle membrane potential or dispersed into endothelial cell sheets for membrane potential recording.Key resultsIntraluminal perfusion of ?-adrenoceptor agonists inhibited endothelium-dependent dilatation to ACh (1 nM-10 ?M) without modifying the associated changes in endothelial cell [Ca²?]i . The inhibitory effect of ?-adrenoceptor agonists was mimicked by direct activation of adenylyl cyclase with forskolin, blocked by the ?-adrenoceptor antagonists propranolol (non-selective), atenolol (??) or the PKA inhibitor KT-5720, but remained unaffected by ICI 118 551 (??) or glibenclamide (ATP-sensitive K? channels channel blocker). Endothelium-dependent hyperpolarization to ACh was also inhibited by ?-adrenoceptor stimulation in both intact arteries and in endothelial cells sheets. Blocking IK(Ca) {with 1 ?M 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34)}, but not SK(Ca) (50 nM apamin) channels prevented ?-adrenoceptor agonists from suppressing either hyperpolarization or vasodilatation to ACh.Conclusions and implicationsIn resistance arteries, endothelial cell ??-adrenoceptors link to inhibit endothelium-dependent hyperpolarization and the resulting vasodilatation to ACh. This effect appears to reflect inhibition of endothelial IK(Ca) channels and may be one consequence of raised circulating catecholamines.

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Murine breast cancer feed arteries are thin-walled with reduced ?1A-adrenoceptor expression and attenuated sympathetic vasocontraction.

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Murine breast cancer feed arteries are thin-walled with reduced α<sub>1A</sub>-adrenoceptor expression and attenuated sympathetic vasocontraction.

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