Thromboxane Analogue U46619 Research Articles

Placental Protein 13: Vasomodulatory Effects on Human Uterine Arteries and Potential Implications for Preeclampsia.

Placental protein 13 (PP13) exhibits a plasma concentration that increases gradually during normal gestation, a process that is disrupted in preeclampsia, which is characterized by elevated vascular resistance, reduced utero-placental blood flow, and intrauterine growth restriction. This study investigated PP13's role in vascular tone regulation and its molecular mechanisms. Uterine and subcutaneous arteries, isolated from both pregnant and non-pregnant women, were precontracted with the thromboxane analogue U46619 and exposed to PP13 using pressurized myography. The molecular mechanisms were further investigated, using specific inhibitors for nitric oxide synthase (L-NAME+LNNA at 10-4 M) and guanylate cyclase (ODQ at 10-5 M). The results showed that PP13 induced vasodilation in uterine arteries, but not in subcutaneous arteries. Additionally, PP13 counteracted U46619-induced vasoconstriction, which is particularly pronounced in pregnancy. Further investigation revealed that PP13's mechanism of action is dependent on the activation of the nitric oxide-cGMP pathway. This study provides novel insights into the vasomodulatory effects of PP13 on human uterine arteries, underscoring its potential role in regulating utero-placental blood flow. These findings suggest that PP13 may be a promising candidate for improving utero-placental blood flow in conditions such as preeclampsia. Further research and clinical studies are warranted to validate PP13's efficacy and safety as a therapeutic agent for managing preeclampsia.

The impact of temperature on vascular function in connection with vascular laser treatment

Pulsed dye lasers are used effectively in the treatment of psoriasis with long remission time and limited side effects. It is, however, not completely understood which biological processes underlie its favorable outcome. Pulsed dye laser treatment at 585-595 nm targets hemoglobin in the blood, inducing local hyperthermia in surrounding blood vessels and adjacent tissues. While the impact of destructive temperatures on blood vessels has been well studied, the effects of lower temperatures on the function of several cell types within the blood vessel wall and its periphery are not known. The aim of our study is to assess the functionality of isolated blood vessels after exposure to moderate hyperthermia (45 to 60°C) by evaluating the function of endothelial cells, smooth muscle cells, and vascular nerves. We measured blood vessel functionality of rat mesenteric arteries (n=19) by measuring vascular contraction and relaxation before and after heating vessels in a wire myograph. To this end, we elicited vascular contraction by addition of either high potassium solution or the thromboxane analogue U46619 to stimulate smooth muscle cells, and electrical field stimulation (EFS) to stimulate nerves. For measurement of endothelium-dependent relaxation, we used methacholine. Each vessel was exposed to one temperature in the range of 45-60°C for 30 seconds and a relative change in functional response after hyperthermia was determined by comparison with the response per stimulus before heating. Non-linear regression was used to fit our dataset to obtain the temperature needed to reduce blood vessel function by 50% (Half maximal effective temperature, ET50). Our findings demonstrate a substantial decrease in relative functional response for all three cell types following exposure to 55°C-60°C. There was no significant difference between the ET50 values of the different cell types, which was between 55.9°C and 56.9°C (P>0.05). Our data show that blood vessel functionality decreases significantly when exposed to temperatures between 55°C-60°C for 30 seconds. The results show functionality of endothelial cells, smooth muscle cells, and vascular nerves is similarly impaired. These results help to understand the biological effects of hyperthermia and may aid in tailoring laser and light strategies for selective photothermolysis that contribute to disease modification of psoriasis after pulsed dye laser treatment.

Abstract 484: Antidiabetic Agent, Canagliflozin, Is A Potent Vasodilator Of The Coronary Microvasculature

Objective: Sodium-glucose cotransporter-2 (SGLT-2) inhibitors are cardioprotective even in the absence of preexisting diabetic microvascular disease. Our group has shown SGLT-2 inhibitor, canagliflozin, increases myocardial perfusion without an attendant increase in angiogenesis. We hypothesize canagliflozin accentuates vasodilation of coronary microvasculature to increase perfusion. Methods: Coronary resistance arterioles (174 ± 60μm in diameter) isolated from Sprague-Dawley rats either receiving daily canagliflozin (15mg/kg, oral gavage) for four weeks (n = 4) or no treatment (n = 4) were studied in vitro in a pressurized (40mmHg), no-flow state using video microscopy. Following preconstriction of microvessels with thromboxane analog U46619 or endothelin-1, concentration response curves to vasodilatory adenosine 5’-diphosphate (ADP), acetylcholine (ACh), and sodium nitroprusside (SNP) were completed. Additionally, nitric-oxide-dependent ADP-induced vasodilation of microvessels isolated from treated rats was measured in the presence of N ω -Nitro-L-arginine methyl ester (L-NAME) 10μM. Results: Canagliflozin treatment was associated with a significant increase in endothelium-dependent vasodilatory response to ADP (p = 0.039) (Figure 1). Pretreatment with L-NAME, an inhibitor of nitric oxide synthase, eliminated this vasodilatory response. Compared to ADP, vasodilatory response to ACh was muted (p = 0.552). Endothelium-independent vasodilatory response to SNP, a nitric oxide donor, strongly trended toward an increase in the treated group (p = 0.099). Conclusions: Canagliflozin improves microvascular vasodilation in an endothelium-dependent and endothelium-independent fashion not yet described. These findings implicate both vascular endothelium and arteriolar smooth muscle cells in altered vasomotor physiology and may, in part, account for the observed increase in myocardial blood flow with canagliflozin treatment.

Phosphodiesterases type 2, 3 and 4 promote vascular tone in mesenteric arteries from rats with heart failure

Phosphodiesterases (PDE) type 3 and 4 promote vasoconstriction by hydrolysing cAMP. In experimental heart failure (HF), PDE3 makes PDE4 redundant in aorta, but it is not known if this occurs in resistance vessels, such as mesenteric artery. As PDE2 is increased in the failing myocardium, its possible role in the vasculature also needs to be addressed. Here, the function of PDE2, PDE3 and PDE4 in rat mesenteric arteries was characterized in experimental HF.Mesenteric arteries were isolated from rats sacrificed 22 weeks after surgical stenosis of the ascending aorta (HF), or Sham surgery. PDE inhibitors were used to probe isoenzyme contributions in enzymatic and isometric tension assays. PDE2 and PDE4 activities, but not PDE3 activity, facilitate contraction produced by the thromboxane analogue U46619 in Sham arteries, while in HF all three isoenzymes contribute to this response. NO synthase inhibition by L-NAME abolished the action of the PDE2 inhibitor. L-NAME eliminated the contribution of PDE4 in HF, but unmasked a contribution for PDE3 in Sham. PDE3 and PDE4 activities attenuated relaxant response to β-adrenergic stimulation in Sham and HF. PDE2 did not participate in cAMP or cGMP-mediated relaxant responses. PDE3 and PDE4 cAMP-hydrolysing activities were smaller in HF mesenteric arteries, while PDE2 activity was scarce in both groups. Endothelial cells and arterial myocytes displayed PDE2 immunolabelling.We highlight that, by contrast with previous observations in aorta, PDE4 participates equally as PDE3 in contracting mesenteric artery in HF. PDE2 activity emerges as a promoter of contractile response that is preserved in HF.

Fetal Sex and Fetal Environment Interact to Alter Diameter, Myogenic Tone, and Contractile Response to Thromboxane Analog in Rat Umbilical Cord Vessels.

Fetal growth needs adequate blood perfusion from both sides of the placenta, on the maternal side through the uterine vessels and on the fetal side through the umbilical cord. In a model of intrauterine growth restriction (IUGR) induced by reduced blood volume expansion, uterine artery remodeling was blunted. The aim of this study is to determine if IUGR and fetus sex alter the functional and mechanical parameters of umbilical cord blood vessels. Pregnant rats were given a low sodium (IUGR) or a control diet for the last 7 days of pregnancy. Umbilical arteries and veins from term (22 day) fetal rats were isolated and set-up in wire myographs. Myogenic tone, diameter, length tension curve and contractile response to thromboxane analog U46619 and serotonin (5-HT) were measured. In arteries from IUGR fetuses, myogenic tone was increased in both sexes while diameter was significantly greater only in male fetuses. In umbilical arteries collected from the control group, the maximal contraction to U46619 was lower in females than males. Compared to the control groups, the maximal response decreased in IUGR male arteries and increased in female ones, thus abolishing the sexual dimorphism observed in the control groups. Reduced contractile response to U46619 was observed in the IUGR vein of both sexes. No difference between groups was observed in response to 5HT in arteries. In conclusion, the change in parameters of the umbilical cord blood vessels in response to a mild insult seems to show adaptation that favors better exchange of deoxygenated and wasted blood from the fetus to the placenta with increased myogenic tone.

Beneficial Changes in Rat Vascular Endocannabinoid System in Primary Hypertension and under Treatment with Chronic Inhibition of Fatty Acid Amide Hydrolase by URB597.

Our study aimed to examine the effects of hypertension and the chronic administration of the fatty acid amide hydrolase (FAAH) inhibitor URB597 on vascular function and the endocannabinoid system in spontaneously hypertensive rats (SHR). Functional studies were performed on small mesenteric G3 arteries (sMA) and aortas isolated from SHR and normotensive Wistar Kyoto rats (WKY) treated with URB597 (1 mg/kg; twice daily for 14 days). In the aortas and sMA of SHR, endocannabinoid levels and cannabinoid CB1 receptor (CB1R) expression were elevated. The CB1R antagonist AM251 diminished the methanandamide-evoked relaxation only in the sMA of SHR and enhanced the vasoconstriction induced by phenylephrine and the thromboxane analog U46619 in sMA in SHR and WKY. In the sMA of SHR, URB597 elevated anandamide levels, improved the endothelium-dependent vasorelaxation to acetylcholine, and in the presence of AM251 reduced the vasoconstriction to phenylephrine and enhanced the vasodilatation to methanandamide, and tended to reduce hypertrophy. In the aortas, URB597 elevated endocannabinoid levels improved the endothelium-dependent vasorelaxation to acetylcholine and decreased CB1R expression. Our study showed that hypertension and chronic administration of URB597 caused local, resistance artery-specific beneficial alterations in the vascular endocannabinoid system, which may bring further advantages for therapeutic application of pharmacological inhibition of FAAH.

NTPDase1 Modulates Smooth Muscle Contraction in Mice Bladder by Regulating Nucleotide Receptor Activation Distinctly in Male and Female.

Nucleotides released by smooth muscle cells (SMCs) and by innervating nerve terminals activate specific P2 receptors and modulate bladder contraction. We hypothesized that cell surface enzymes regulate SMC contraction in mice bladder by controlling the concentration of nucleotides. We showed by immunohistochemistry, enzymatic histochemistry, and biochemical activities that nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) and ecto-5′-nucleotidase were the major ectonucleotidases expressed by SMCs in the bladder. RT-qPCR revealed that, among the nucleotide receptors, there was higher expression of P2X1, P2Y1, and P2Y6 receptors. Ex vivo, nucleotides induced a more potent contraction of bladder strips isolated from NTPDase1 deficient (Entpd1−/−) mice compared to wild type controls. The strongest responses were obtained with uridine 5′-triphosphate (UTP) and uridine 5′-diphosphate (UDP), suggesting the involvement of P2Y6 receptors, which was confirmed with P2ry6−/− bladder strips. Interestingly, this response was reduced in female bladders. Our results also suggest the participation of P2X1, P2Y2 and/or P2Y4, and P2Y12 in these contractions. A reduced response to the thromboxane analogue U46619 was also observed in wild type, Entpd1−/−, and P2ry6−/− female bladders showing another difference due to sex. In summary, NTPDase1 modulates the activation of nucleotide receptors in mouse bladder SMCs, and contractions induced by P2Y6 receptor activation were weaker in female bladders.

Abstract 15585: Sex Dimorphism in Prostaglandins’ Role in Thromboxane-dependent Contraction in the Rat Middle Cerebral Artery

Introduction: Recent evidences suggest that sex and estrogen can influence cerebrovascular reactivity. We investigated the role of prostanoids and the estrogen receptor (ER) ex vivo in isolated middle cerebral arteries from male (M-MCA) and female (F-MCA) rats. Methods: MCA segments isolated from 20-week-old male and female Sprague-Dawley rats were mounted on a wire Multi Myograph (DMT) to measure isometric tone. Acetylcholine (ACh) was used to assess endothelium integrity. Concentration response curves to the thromboxane analog U46619 (U4, 10 -11 -10 -5 M) were performed in arteries intact or pre-treated with indomethacin (I,10 -5 M) or the ER antagonist G15 (G, 10 -6 M). Data were acquired with Powerlab 8 (ADInstruments) and recorded with LabChart v8 (ADInstruments). Response to K + was expressed in mN/mm whereas U4 maximal contraction as percent of maximal response to 75mM K + (%K MAX ) and sensitivity as pD 2 (-LogEC 50 ). Results: M-MCA (n=10) and F-MCA (n=13) displayed similar optimal diameters (M vs. F; 214±12 vs. 218±5 μm, p>0.05) and ACh-dependent relaxation (M vs. F; 71±11 vs. 78±10 % pre-constricted tone; 7.3±0.4 vs. 7.2±0.2, p>0.05). A greater contraction to K + was observed in M-MCA (M vs. F; 1±0.2 vs. 0.6±0.1 mN/mm, p<0.05). M- and F-MCA showed similar maximal contraction (M vs. F; 116±6 vs. 109±10 %K MAX ) and sensitivity (M vs. F; 7.2±0.2 vs. 7.3±0.2) to U4. Pre-incubation with Indomethacin lowered maximal response and sensitivity (M vs. M-I; 116±6 vs. 97±5%K MAX, p<0.05; 7.2±0.2 vs. 6.8±0.1, p<0.05) to U4 in M-MCA, with no effect on F-MCA (F vs. F-I; 109±9 vs. 113±7%K MAX ; 7.3±0.2 vs. 7.3±0.2). Pre-treatment with G15 increased U4 sensitivity in M-MCA (M vs. M-G; 7.3±0.2 vs. 7.8±0.2, p<0.05), with no effect in F-MCA (F vs. F-G; 7.3±0.2 vs. 7.4±0.3). Conclusions: Our data demonstrate sex differences in thromboxane-dependent contraction of MCA in middle-aged rats. Hormonal changes during the estrous cycle may contribute to a greater variability of F-MCA responses.

Flow Cytometric Monitoring of Dynamic Cytosolic Calcium, Sodium, and Potassium Fluxes Following Platelet Activation.

The interaction of platelet agonists with their respective membrane receptors triggers intracellular signaling, among which cytosolic ion fluxes play an important role in activation processes. While the key contribution of intercellular free calcium is accepted, sodium and potassium roles in platelet activation have been less investigated in recent studies. Here, we implemented a novel flow-cytometric method to monitor over time cytosolic free calcium, sodium, and potassium ion fluxes upon platelet activation and we demonstrate the feasibility of real-time visualization of ion kinetics, in particular with a focus on sodium and potassium. Platelets were loaded with selective ion indicators, Fluo-3 (Ca2+ ), ION NaTRIUM Green-2 (Na+ ), and ION Potassium Green-2 (K+ ). Fluorescence was monitored by flow cytometry. After measurement of a stable baseline, platelets were activated and ion indicator fluorescence was acquired over time, up to 10 min. Platelets were activated with either thromboxane analogue U46619, ADP, thrombin, TRAP6 (PAR-1 agonist), AYPGKF (PAR-4 agonist), convulxin (collagen receptor GPVI agonist), or combinations thereof. We evaluated preanalytical parameters (in particular dye loading time and concentration) to implement an accurate method. Subsequently, we characterized cytosolic calcium, sodium, and potassium kinetics in response to platelet agonists. We observed different patterns of agonist synergism. In conclusion, the present work highlights the use of cytosolic ion monitoring by flow cytometry to investigate characteristic calcium, sodium, and potassium mobilization patterns following platelet activation. This easy technique opens a new way to analyze signaling in different platelet subpopulations and it should prove useful for investigating platelet pathophysiology. © 2020 International Society for Advancement of Cytometry.

Inhibition of Female and Male Human Detrusor Smooth Muscle Contraction by the Rac Inhibitors EHT1864 and NSC23766

IntroductionLower urinary tract symptoms (LUTS) due to overactive bladder (OAB) are caused by spontaneous detrusor contractions. Medical treatment with muscarinic receptor antagonists or β3-adrenoceptor agonists aims to inhibit detrusor contractions, but overall results are unsatisfactory. Consequently, improved understanding of bladder smooth muscle contraction and identification of novel compounds for its inhibition are needed to develop alternative options. A role of the GTPase Rac1 for smooth muscle contraction has been reported from the prostate, but is unknown in the human detrusor. Here, we examined effects of the Rac inhibitors NSC23766, which may also antagonize muscarinic receptors, and EHT1864 on contraction of human detrusor tissues.MethodsFemale and male human detrusor tissues were obtained from radical cystectomy. Effects of NSC23766 (100 µM) and EHT1864 (100 µM) on detrusor contractions were studied in an organ bath.ResultsElectric field stimulation induced frequency-dependent contractions of detrusor tissues, which were inhibited by NSC23766 and EHT1864. Carbachol induced concentration-dependent contractions. Concentration response curves for carbachol were shifted to the right by NSC23766, reflected by increased EC50 values, but unchanged Emax values. EHT1864 reduced carbachol-induced contractions, resulting in reduced Emax values for carbachol. The thromboxane analog U46619 induced concentration-dependent contractions, which remained unchanged by NSC23766, but were reduced by EHT1864.ConclusionsNSC23766 and EHT1864 inhibit female and male human detrusor contractions. NSC23766, but not EHT1864 competitively antagonizes muscarinic receptors. In addition to neurogenic and cholinergic contractions, EHT1864 inhibits thromboxane A2-induced detrusor contractions. The latter may be promising, as the origin of spontaneous detrusor contractions in OAB is noncholinergic. In vivo, both compounds may improve OAB-related LUTS.

Platelet CD34 Expression and a Congenital Collar Bone Malformation Associated with a Partial CBFB Deletion in a Case with a Bleeding Disorder

Mutations in the transcription factor Growth Factor Independence 1B (GFI1B) and Runt-related transcription factor 1 (RUNX1) are causal to familial bleeding disorders. Mutant RUNX1 and GFI1B affect megakarocyte development, resulting in decreased numbers of platelets with significantly reduced α-granules and platelets that express the hematopoietic stem and progenitor cell marker CD34. A case that presented in our clinic with a bleeding disorder (Tosetto bleeding score of 11) was diagnosed with a δ-granule secretion defect. Platelet light transmission aggregometry revealed an aggregation defect upon stimulation with epinephrine and low concentrations of collagen and ADP. ATP release from δ-granules was normal after stimulation with a high concentration of collagen, however, no ATP was released upon stimulation with epinephrine and low levels were measured after stimulation with the thromboxane analogue U46619. Whole mount electron microscopy showed increased numbers of δ-granules in platelets derived from the index case compared to controls (average 5.84 δ-granules vs 3.66, per platelet respectively). To identify a genetic cause for the disease, a bleeding disorder gene panel was screened following whole exome sequencing. No disease-causing variants were called. Open exome analysis detected a ~100Kb heterozygous chromosome 16q22.1 deletion that was confirmed by SNP array. The observed deletion covers nine genes, including the last exon of CBFB. To date, none of the deleted genes have been implicated in inherited bleeding or platelet disorders. However, conditional knockout of Cbfb in mice results in a plethora of hematopoietic abnormalities including disturbed megakaryopoiesis and thrombocytopenia. CBFB is part of a heterodimeric transcription factor complex with either RUNX1, -2 or -3. Because of the CBFB-RUNX1 interaction and the fact that bleeding disorders caused by RUNX1 mutations result in platelet CD34 expression, we tested the platelets of the case reported here. Using flow cytometry, we clearly detected platelet CD34 expression compared to healthy controls, albeit to a lower level compared to RUNX1 and GFI1B affected cases. Mutations in RUNX2 may cause cleidocranial dysplasia and entire CBFB deletions have been reported in rare cases with congenital bone abnormalities. The medical record of the case presented here indicated that she had undergone surgery at the age of 13 for a congenital malformed collar bone. These data suggest a relation between the 16q/CBFB deletion and the observed abnormalities. To address this, we determined whether the abnormalities segregated with the 16q deletion. The mother and son of the index case had no history of clinical bleedings, no signs of bone malformations and did not have the 16q deletion. The father who was deceased was reported by the family not to have a bleeding propensity nor bone abnormalities. Platelets from the son did not express CD34. Remarkably, platelets from the son showed an aggregation defect upon stimulation with epinephrine and ADP, similar to the index patient. No ATP was released from δ-granules following stimulation with epinephrine. Thus, the 16q deletion associates with platelet CD34 expression and a congenital bone abnormality but is not responsible for the δ -granule secretion defect as the latter was observed in the son who did not have the 16q deletion, nor bleedings. Defective downmodulation of CD34 during platelet formation in the index case reported here suggests abnormal megakaryocyte development that might contribute in a multifactorial manner to bleedings. Recently, we reported CD34 expression on platelets from patients with rare GFI1B variants that may predispose to a bleeding propensity, but do not cause bleedings on their own. Thus, it will be important to determine whether the 16q deletion (and C-terminal truncation of CBFB) reported here affects megakaryocyte development and if so, contributes to the bleeding disorder in the index case. In contrast to Cbfb null mice, Cbfb heterozygous mice do not exhibit discernable hematopoietic phenotypes. Thus, if the C-terminal CBFB truncation contributes to the disease characteristics, it is likely that this occurs through a dominant-negative mechanism rather than haploinsufficiency. The findings reported here warrant further studies into the role of 16q (and CBFB) deletions in megakaryocyte development. Disclosures No relevant conflicts of interest to declare.

Hyperglycemia Enhances Constriction of Retinal Venules via Activation of the Reverse-Mode Sodium-Calcium Exchanger.

Diabetes is associated with hyperglycemia and impairment of retinal microvascular function. However, the impact of hyperglycemia on retinal venular constriction remains unknown. We examined retinal venular responsiveness to endogenous vasoconstrictors and the contribution of the reverse-mode sodium-calcium exchanger (NCX) to these responses during hyperglycemia. Retinal venules were isolated from pigs with streptozocin-induced diabetes (2 weeks, in vivo hyperglycemia) and age-matched control pigs for vasoreactivity and molecular studies. For in vitro hyperglycemia, vessels from euglycemic pigs were exposed to high glucose (25 mmol/L) for 2 h, and 5 mmol/L glucose served as the control. Constrictions of venules from euglycemic pigs to endothelin-1 (ET-1), thromboxane analog U46619, and norepinephrine were mediated by ETA, thromboxane, and α2-adrenergic receptors, respectively, and were insensitive to reverse-mode NCX blockade (KB-R7943). In vivo hyperglycemia enhanced these vasoconstrictions without altering respective receptor mRNA expression. Similarly, in vitro hyperglycemia augmented venular constrictions. Enhanced vasoconstrictions during hyperglycemia were prevented by KB-R7943, while mRNA expression of venular NCX isoforms was unaltered. In vivo hyperglycemia increased vitreous levels of ET-1 but not thromboxane B2 In conclusion, both in vitro and in vivo hyperglycemia enhance retinal venular responses to endogenous vasoconstrictors by activating reverse-mode NCX. Therapies targeting this vascular molecule may alleviate retinal complications during diabetes.

Increased platelet reactivity and platelet–leukocyte aggregation after elective coronary bypass surgery

Inflammatory mechanisms are activated, and thrombotic complications occur during the initial months after coronary artery bypass grafting (CABG). Therefore, changes over time of platelet activation and platelet–leukocyte interactions after CABG are of interest. Whole-blood flow cytometry was performed before, and 4–6 days, one month, and three months after elective CABG in 54 men with stable coronary artery disease treated with acetylsalicylic acid (ASA). Single platelets and platelet–leukocyte aggregates (PLAs) among monocytes (P-Mon), neutrophils (P-Neu), and lymphocytes (P-Lym) were studied without and with stimulation by submaximal concentrations of ADP, thrombin, and the thromboxane analog U46619. White blood cell counts were increased during the initial postoperative course, and platelet counts were increased after one month. Platelet P-selectin expression was significantly enhanced at one month when stimulated by thrombin and U46619 and at three months with ADP and thrombin. All PLAs subtypes were increased at one month without stimulation in vitro. P-Mon and P-Neu stimulated by ADP, thrombin, or U46619 were significantly increased one month after the operation but decreased compared to baseline at three months. Agonist stimulated P-Lyms were increased at one month and remained increased at three months after ADP stimulation. There was significant platelet activation and formation of PLAs unstimulated and after agonist stimulation by ADP, thrombin, and a thromboxane analog after CABG in patients with stable coronary artery disease irrespective of ASA treatment. Changes observed up to three months after CABG support further studies of the clinical implications of protracted increases in platelet activation and platelet–leukocyte interactions.

Murine breast cancer feed arteries are thin-walled with reduced \u03b11A-adrenoceptor expression and attenuated sympathetic vasocontraction

BackgroundPerfusion 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.MethodsWe 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 α1-adrenoceptor antagonist prazosin.ResultsBreast 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 α1-adrenoceptors and messenger RNA levels for α1A-adrenoceptors are lower in breast cancer arteries than control arteries. Sympathetic vasocontraction elicited by electrical field stimulation is inhibited by α1-adrenoceptor blockade and reduced in breast cancer feed arteries compared to control arteries.ConclusionThinner media and lower α1-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.

The β2 agonist terbutaline specifically decreases pulmonary arterial pressure under normoxia and hypoxia via α adrenoceptor antagonism.

Pulmonary hypertension is a severe, incurable disease with a poor prognosis. Although treatment regimens have improved during the last 2 decades, current pharmacologic strategies are limited and focus on the modulation of only a few pathways related to endothelin, NO, and prostacyclin signaling. Therefore, the identification of novel molecular targets is urgently needed. We found that the β2 adrenoceptor (AR) agonists terbutaline (TER) and salbutamol induced a dose-dependent vasorelaxation in large pulmonary arteries but not aortas of mouse. This effect was found to be independent of β ARs and the endothelium but was determined by the type of the preconstrictor. Vasodilation by β2 AR agonists occurred after pretreatment of pulmonary arteries with phenylephrine and serotonin, both agonists of α1 ARs, but was absent after preconstriction with the thromboxane analog U46619. These data indicated α-adrenolytic activity of β2 AR agonists, which was confirmed by a right shift of the phenylephrine dose-response curve by TER. This effect was physiologically relevant because TER also relaxed small intrapulmonary arteries in lung slices and diminished pulmonary arterial pressure in an isolated perfused lung model under normoxia and hypoxia. Finally, TER applied as an aerosol also selectively decreased pulmonary arterial pressure without effects on systemic blood pressure and heart rate in mouse in vivo. Thus, β2 AR agonists display α-adrenolytic activity in pulmonary arteries ex vivo and in vivo, and may provide a novel option to reduce pulmonary arterial pressure in pulmonary hypertension.-Neumann, V., Knies, R., Seidinger, A., Simon, A., Lorenz, K., Matthey, M., Breuer, J., Wenzel, D. The β2 agonist terbutaline specifically decreases pulmonary arterial pressure under normoxia and hypoxia via α adrenoceptor antagonism.

Abstract P103: Anti-hypertensive Mechanisms of Action of G q/11 Inhibitor Ligands

Chronic blockade of individual G protein coupled receptors (GPCRs) has proven to be inadequate strategy for managing hypertension partly because the subcellular heterotrimeric G proteins that propagate intracellular signaling can simultaneously couple to several other vasopressor receptors. Whether blood pressure can better be controlled by directly targeting G proteins has not been thoroughly investigated due to paucity of selective, cell-permeable inhibitors. Here, we tested whether chemical inhibition of Gq/11 proteins in vivo and ex vivo using recently discovered small-molecule inhibitor ligands, YM-254890 (YM), FR-900359 (FR) and WU-07047 (WU) is sufficient to reverse hypertension in mice. Using ex vivo vessel reactivity assay, we found that Gq/11 inhibitors markedly reduced vasoconstriction evoked with phenylephrine (PE), vasopressin, endothelin-1, and the thromboxane analog U-46619. Blockade of PE-induced contractility by the Gq/11 inhibitors showed the following rank-order potency: FR LogIC50 -0.008 ± 0 > YM LogIC50 -0.49 ± 0 > WU LogIC50 -64.95 ± 6.4. YM and WU but not FR inhibited PE-induced vasoconstriction through G protein-dependent and independent pathways by blocking L-type calcium channel-mediated Ca 2+ influx. Acute subcutaneous injection of FR and YM (0.3 mg/kg, s.c.) in normotensive and N ω -Nitro-L-arginine methyl ester (L-NAME) hypertension mice elicited marked hypotension, which was more severe (ΔSBP = -25 ± 2.7 vs. ΔSBP = -21 ± 2.2 mmHg) and long lasting (FR t1/2 ≅ 12 hr vs. YM t1/2 ≅ 4 hr) after the injection of FR relative to YM. In DOCA-salt hypertension mice, chronic injection of FR (0.3 mg/kg, s.c., daily for seven days) reversed hypertension (vehicle SBP: 149 ± 5 vs. FR SBP: 117 ± 7 mmHg) and sustained blood pressure reduction several days after terminating the injection regimen (DOCA SBP: 141 ± 2 vs. SBP 5 days post FR: 128 ± 5 mmHg). Our results together support the hypothesis that increased Gq/11 activity in blood pressure-regulating organs is involved in the pathogenesis of hypertension, and that direct systemic blockade of Gq/11 reverses hypertension. The findings provide clear evidence for targeting Gq/11 in the cardiovascular system as an effective therapy for treating hypertension.

Rat retinal vasomotion assessed by laser speckle imaging.

Vasomotion is spontaneous or induced rhythmic changes in vascular tone or vessel diameter that lead to rhythmic changes in flow. While the vascular research community debates the physiological and pathophysiological consequence of vasomotion, there is a great need for experimental techniques that can address the role and dynamical properties of vasomotion in vivo. We apply laser speckle imaging to study spontaneous and drug induced vasomotion in retinal network of anesthetized rats. The results reveal a wide variety of dynamical patterns. Wavelet-based analysis shows that (i) spontaneous vasomotion occurs in anesthetized animals and (ii) vasomotion can be initiated by systemic administration of the thromboxane analogue U-46619 and the nitric-oxide donor S-nitroso-acetylDL-penicillamine (SNAP). Although these drugs activate different cellular pathways responsible for vasomotion, our approach can track the dynamical changes they cause.

Inhibition of vascular smooth muscle inward-rectifier K+ channels restores myogenic tone in mouse urinary bladder arterioles.

Prolonged decreases in urinary bladder blood flow are linked to overactive and underactive bladder pathologies. However, the mechanisms regulating bladder vascular reactivity are largely unknown. To investigate these mechanisms, we examined myogenic and vasoactive properties of mouse bladder feed arterioles (BFAs). Unlike similar-sized arterioles from other vascular beds, BFAs failed to constrict in response to increases in intraluminal pressure (5-80 mmHg). Consistent with this lack of myogenic tone, arteriolar smooth muscle cell membrane potential was hyperpolarized (-72.8 ± 1.4 mV) at 20 mmHg and unaffected by increasing pressure to 80 mmHg (-74.3 ± 2.2 mV). In contrast, BFAs constricted to the thromboxane analog U-46619 (100 nM), the adrenergic agonist phenylephrine (10 µM), and KCl (60 mM). Inhibition of nitric oxide synthase or intermediate- and small-conductance Ca2+-activated K+ channels did not alter arteriolar diameter, indicating that the dilated state of BFAs is not attributable to overactive endothelium-dependent dilatory influences. Myocytes isolated from BFAs exhibited BaCl2 (100 µM)-sensitive K+ currents consistent with strong inward-rectifier K+ (KIR) channels. Notably, block of these KIR channels "restored" pressure-induced constriction and membrane depolarization. This suggests that these channels, in part, account for hyperpolarization and associated absence of tone in BFAs. Furthermore, smooth muscle-specific knockout of KIR2.1 caused significant myogenic tone to develop at physiological pressures. This suggests that 1) the regulation of vascular tone in the bladder is independent of pressure, insofar as pressure-induced depolarizing conductances cannot overcome KIR2.1-mediated hyperpolarization; and 2) maintenance of bladder blood flow during bladder filling is likely controlled by neurohumoral influences.

Non-Adrenergic, Tamsulosin-Insensitive Smooth Muscle Contraction is Sufficient to Replace α1 -Adrenergic Tension in the Human Prostate.

Lower urinary tract symptoms (LUTS) suggestive of benign prostatic hyperplasia may be caused by prostate smooth muscle contraction. Although α1 -blockers may improve symptoms by prostate smooth muscle relaxation, their efficacy is limited. This may be explained by non-adrenergic mediators causing contraction in parallel to α1 -adrenoceptors. However, little is known about the relevance and cooperative actions of non-adrenergic mediators in the prostate. Prostate tissues were obtained from radical prostatectomy (n = 127 patients). Contractile responses were studied in an organ bath. Endothelin-1 and noradrenaline induced contractions of similar magnitude (116 ± 23 and 117 ± 18% of KCl-induced contractions). Endothelin-2- and -3-induced maximum contractions of 63 ± 8.6 and 71 ± 19% of KCl, while contractions by the thromboxane analog U46619 amounted up to 63 ± 9.4%. Dopamine-induced contractions averaged to 22 ± 4.5% of KCl, while maximum contractions by serotonin, histamine, and carbachol stayed below 10% of KCl-induced. While noradrenaline-induced contractions were inhibited by tamsulosin (300 nM), endothelin-1-, -2-, or -3-induced contraction were not. No additive effects were observed if endothelins and noradrenaline were applied consecutively to the same samples. If endothelin-1 was applied after U46619, resulting tension (172 ± 43% of KCl) significantly exceeded noradrenaline-induced contraction. Tensions following combined application of endothelin-2 or -3 with U46619 stayed below noradrenaline-induced contractions. Tension following combined application of all three endothelins with U46619 resembled maximum noradrenaline-induced tone. Contractions following concomitant confrontation of human prostate tissue with noradrenaline and endothelin-1 are not additive. Endothelin-1 is sufficient to induce a smooth muscle tone resembling that of noradrenaline. This may replace lacking α1 -adrenergic tone under therapy with α1 -blockers, explaining the limited efficacy of α1 -blockers in LUTS treatment. Contractions by thromboxane and endothelin-1 may be additive, and may exceed α1 -adrenergic tone. Prostate 77:697-707, 2017. © 2017 Wiley Periodicals, Inc.

Search for an animal model to investigate selective pulmonary vasodilation.

Pulmonary arterial hypertension is a life-threatening disease with a poor prognosis. Oral treatment with vasodilators is often limited by systemic hypotension. Inhalation of vasodilators offers the opportunity for selective pulmonary vasodilation. Testing selective pulmonary vasodilation by inhaled nitric oxide or alternative substances in animal models requires an increased pulmonary vascular tone. The aim of this study was to identify animal models that are suitable for investigating selective pulmonary vasodilation. To do so, a haemodynamic stable pulmonary hypertension was initiated, with a 30 min duration deemed to be a sufficient time interval before and after a possible intervention. In anaesthetized and mechanically-ventilated Sprague-Dawley rats pulmonary hypertension was induced either by acute hypoxia due to reduction of the inspired oxygen fraction from 0.21 to 0.1 ( n = 6), a fixed infusion rate of the thromboxane analogue U46619 (240 ng/min; n = 6) or a monocrotaline injection (MCT; 60 mg/kg applied 23 days before the investigation; n = 7). The animals were instrumented to measure right ventricular and systemic arterial pressures. Acute hypoxia caused a short, and only transient, increase of pulmonary artery pressure as well as profound systemic hypotension which suggested haemodynamic instability. U46619 infusion induced variable changes in the pulmonary and systemic vascular tone without sufficient stabilization within 30 min. MCT provoked sustained pulmonary hypertension with normal systemic pressure values and inhalation of nitric oxide caused selective pulmonary vasodilation. In conclusion, out of the three examined rat animal models only MCT-induced pulmonary hypertension is a solid and reliable model for investigating selective pulmonary vasodilation.