Renal Sympathetic Hyperactivity in Diabetes Is Modulated by 5-HT1D Receptor Activation via NO Pathway.

Renal vasculature, which is highly innervated by sympathetic fibers, contributes to cardiovascular homeostasis. This renal sympathetic outflow is inhibited by 5-HT in normoglycaemic rats. Considering that diabetes induces cardiovascular complications, we aimed to determine whether diabetic state modifies noradrenergic input at renal level and its serotonergic modulation in rats. Alloxan diabetic rats were anaesthetized (pentobarbital; 60 mg/kg i.p.) and prepared for in situ autoperfusion of the left kidney to continuously measure systemic blood pressure (SBP), heart rate (HR), and renal perfusion pressure (RPP). Electrical stimulation of renal sympathetic outflow induces frequency-dependent increases (Δ) in RPP (23.9 ± 2.1, 59.5 ± 1.9, and 80.5 ± 3.5 mm Hg at 2, 4, and 6 Hz, respectively), which were higher than in normoglycaemic rats, without modifying HR or SBP. Intraarterial bolus of 5-HT and 5-CT (5-HT1/5/7 agonist) reduced electrically induced ΔRPP. Only L-694,247 (5-HT1D agonist) reproduced 5-CT inhibition on sympathetic-induced vasoconstrictions, whereas it did not modify exogenous noradrenaline-induced ΔRPP. 5-CT inhibition was exclusively abolished by i.v. bolus of LY310762 (5-HT1D antagonist). An inhibitor of guanylyl cyclase, ODQ (i.v.), completely reversed the L-694,247 inhibitory effect. In conclusion, diabetes induces an enhancement in sympathetic-induced vasopressor responses at the renal level. Prejunctional 5-HT1D receptors, via the nitric oxide pathway, inhibit noradrenergic-induced vasoconstrictions in diabetic rats.

Fluoxetine Treatment Decreases Cardiac Vagal Input and Alters the Serotonergic Modulation of the Parasympathetic Outflow in Diabetic Rats.

Comorbid diabetes and depression constitutes a major health problem, worsening associated cardiovascular diseases. Fluoxetine's (antidepressant) role on cardiac diabetic complications remains unknown. We determined whether fluoxetine modifies cardiac vagal input and its serotonergic modulation in male Wistar diabetic rats. Diabetes was induced by alloxan and maintained for 28 days. Fluoxetine was administered the last 14 days (10 mg/kg/day; p.o). Bradycardia was obtained by vagal stimulation (3, 6 and 9 Hz) or i.v. acetylcholine administrations (1, 5 and 10 µg/kg). Fluoxetine treatment diminished vagally-induced bradycardia. Administration of 5-HT originated a dual action on the bradycardia, augmenting it at low doses and diminishing it at high doses, reproduced by 5-CT (5-HT1/7 agonist). 5-CT did not alter the bradycardia induced by exogenous acetylcholine. Decrease of the vagally-induced bradycardia evoked by high doses of 5-HT and 5-CT was reproduced by L-694,247 (5-HT1D agonist) and blocked by prior administration of LY310762 (5-HT1D antagonist). Enhancement of the electrical-induced bradycardia by 5-CT (10 µg/kg) was abolished by pretreatment with SB269970 (5-HT7 receptor antagonist). Thus, oral fluoxetine treatment originates a decrease in cardiac cholinergic activity and changes 5-HT modulation of bradycardic responses in diabetes: prejunctional 5-HT7 receptors augment cholinergic-evoked bradycardic responses, whereas prejunctional 5-HT1D receptors inhibit vagally-induced bradycardia.

Vascular sympathetic neurotransmission and its serotonergic regulation are modified by chronic fluoxetine treatment.

Given the interconnection between depressive and cardiovascular disorders, we investigated whether antidepressant treatment (fluoxetine) modifies the serotonergic influence on rat vascular noradrenergic outflow. Twelve-week-old male Wistar rats received fluoxetine treatment (10 mg/kg/day; p.o.) for 14 days; then, they were pithed and prepared for sympathetic stimulation. Vasopressor responses were obtained by electrical stimulation of the sympathetic outflow (0.1, 0.5, 1, and 5 Hz) or i.v. noradrenaline (NA; 0.01, 0.05, 0.1, and 0.5 µg/kg). In fluoxetine-treated group, the electrical-induced vasoconstrictions were lower compared to non-treated rats. Intravenous infusion of 5-HT (10 µg/kg/min) inhibited the sympathetically-induced vasoconstrictions. Only 5-CT, 8-OH-DPAT and L-694,247 (5-HT1/7, 5-HT1A and 5-HT1D agonists, respectively) mimicked 5-HT-induced inhibition, while α-methyl-5-HT (5-HT2 agonist) increased the vasopressor responses. The inhibitory effect of 5-HT was: a) no modified by SB269970 (5-HT7 antagonist); b) abolished by WAY-100,635 (5-HT1A antagonist) plus LY310762 (5-HT1D antagonist); and c) potentiated by ritanserin (5-HT2A receptor antagonist). The vasoconstrictions induced by exogenous NA were not modified by 5-CT but were increased by α-methyl-5-HT. Our results suggest that fluoxetine treatment decreases NA release at vascular level and changes 5-HT modulation on rat vascular noradrenergic neurotransmission, inducing sympatho-inhibition via prejunctional 5-HT1A/1D receptors, and sympatho-potentiation via pre and/or postjunctional 5-HT2A receptors.

Dopamine D4 receptor subtype activation reduces the rat cardiac parasympathetic discharge.

The dopaminergic system influences the heart rhythm by inhibiting the rat cardiac sympathetic and parasympathetic neurotransmissions through activation of D2-like receptors (encompassing the D2, D3, and D4 subtypes). Whereas D2 receptor subtype activation results in cardiac sympatho-inhibition, the dopamine receptor subtypes involved in rat cardiac vago-inhibition remain unknown. Hence, this study investigated the specific functional role of the D2-like receptor subtypes (D2, D3, and/or D4) inhibiting the rat heart cholinergic drive. For this purpose, male Wistar rats were pithed and prepared for cardiac vagal stimulation. Bradycardic responses were obtained by electrical stimulation of vagal fibres (3, 6, 9 Hz; n = 100) or i.v. acetylcholine (ACh; 1, 5, 10 µg/kg; n = 15). Expression of D2, D3, and D4 receptors was studied in left and right atrium samples by PCR (n = 4). Intravenous injections of quinpirole (D2-like agonist; 1-30 µg/kg), but not of SFK-38393 (D1-like agonist; 1-30 µg/kg), dose-dependently inhibited the vagally induced bradycardia. The vago-inhibition induced by quinpirole (which failed to affect the bradycardia to i.v. ACh) was unchanged after i.v. injections of the antagonists L-741,626 (D2; 100 µg/kg) or SB-277011-A (D3; 100 µg/kg), but it was abolished by L-745,870 (D4; 100 µg/kg). mRNA levels of D2, D3, and D4 receptor subtype were detected in the left and right rat atria. Our results suggest that the quinpirole-induced vagolytic effect involves prejunctional D4 receptor subtypes, located in the left and right atria. This provides new evidence on the relevance of D4 receptor modulating the heart parasympathetic control.

Tumor suppressive function of E2F-1 on PTEN-induced serrated colorectal carcinogenesis.

Many human cancers present Phosphatase and tensin homolog (PTEN) deficiency and between 20 and 30% of colorectal tumors show PTEN loss. The transcription factor, E2 promoter binding factor 1 (E2F-1), exhibits tumor promoter or suppressive functions depending on cellular type and tissue context, but its role in the progression and development of colorectal carcinogenesis was largely unknown. Here, using a tamoxifen-inducible PTEN knockout mouse model, we have demonstrated that loss of PTEN leads to the development of colorectal tumorigenesis through the serrated pathway. Next, we studied PTEN loss-driven colorectal lesions in the context of E2F-1 deficiency in vivo. Our results revealed that monoallelic and biallelic absence of E2F-1 led to an increased incidence and progression of serrated tumorigenesis induced by PTEN loss. Finally, we investigated the mechanisms by which double PTEN/E2F-1 deficiency leads to enhanced tumorigenesis. We found that colorectal tumors from PTEN/E2F-1 double knockout mice and the human colorectal carcinoma cell line HT29 with shRNA-mediated downregulation of PTEN and E2F-1 exhibit hyperactivation of the RAS-MAPK pathway, accumulation of DNA damage and resistance to apoptosis. To date, this is the first preclinical study evaluating the effect of genetic deletion of E2F-1 in colorectal malignancies driven by PTEN deficiency. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Blockade of 5-HT2 receptors with sarpogrelate uncovers 5-HT7 receptors inhibiting the tachycardic sympathetic drive in pithed rats.

Our group has previously shown in pithed rats that the cardiac sympathetic drive, which produces tachycardic responses, is inhibited by 5-HT via the activation of prejunctional 5-HT1B/1D/5 receptors. Interestingly, when 5-HT2 receptors are chronically blocked with sarpogrelate, the additional role of cardiac sympatho-inhibitory 5-HT1F receptors is unmasked. Although 5-HT2 receptors mediate tachycardia in rats, and the chronic blockade of 5-HT2 receptors unmasked 5-HT7 receptors mediating cardiac vagal inhibition, the role of 5-HT7 receptors in the modulation of the cardiac sympathetic tone remains virtually unexplored. On this basis, male Wistar rats were pretreated during 14 days with sarpogrelate (a 5-HT2 receptor antagonist) in drinking water (30 mg/kg/day; sarpogrelate-pretreated group) or equivalent volumes of drinking water (control group). Subsequently, the rats were pithed to produce increases in heart rate by either electrical preganglionic spinal (C7 -T1 ) stimulation of the cardiac sympathetic drive or iv administration of exogenous noradrenaline. The iv continuous infusion of AS-19 (a 5-HT7 receptor agonist; 10 µg/kg/min) (i) inhibited the tachycardic responses to sympathetic stimulation, but not those to exogenous noradrenaline only in sarpogrelate-pretreated rats. This inhibition was completely reversed by SB258719 (a selective 5-HT7 receptor antagonist; 1 mg/kg, iv) or glibenclamide (an ATP-sensitive K+ channel blocker; 20 mg/kg, iv). These results suggest that chronic 5-HT2 receptor blockade uncovers a cardiac sympatho-inhibitory mechanism mediated by 5-HT7 receptors, involving a hyperpolarization due to the opening of ATP-sensitive K+ channels. Thus, these findings support the role of 5-HT7 receptors in the modulation of the cardiac sympathetic neurotransmission.

Fluoxetine oral treatment discloses 5-HT1D receptor as vagoinhibitor of the cardiac cholinergic neurotransmission in rat.

Although depression and cardiovascular diseases are related, the role of antidepressants such as fluoxetine (increasing serotonin levels) within cardiac regulation remains unclear. We aimed to determine whether fluoxetine modifies the pharmacological profile of serotonergic influence on vagal cardiac outflow. Rats were treated with fluoxetine (10 mg/kg per day; p.o.) for 14 days or equivalent volumes of drinking water (control group); then, they were pithed and prepared for vagal stimulation. Bradycardic responses were obtained by electrical stimulation of the vagal fibers (3, 6, and 9 Hz) or i.v. acetylcholine (ACh; 1, 5, and 10 µg/kg). The i.v. administration of 5-hydroxytryptamine (5-HT; 10 and 50 µg/kg) inhibited the vagally induced bradycardia. 5-CT (5-HT1/7 agonist) and L-694,247 (5-HT1D agonist) mimicked the serotonin inhibitory effect while α-methyl-5-HT (5-HT2 agonist) was devoid of any action. SB269970 (5-HT7 antagonist) did not abolish 5-CT inhibitory action on the electrically induced bradycardia. Pretreatment with LY310762 (5-HT1D antagonist) blocked the effects induced by L-694,247 and 5-CT. 5-HT and 5-CT failed to modify the bradycardia induced by exogenous ACh. Our outcomes suggest that fluoxetine treatment modifies 5-HT modulation on heart parasympathetic neurotransmission in rats, evoking inhibition of the bradycardia via prejunctional 5-HT1D in pithed rats.

5-HT modulates the rat mesenteric vasopressor outflow by 5-HT1D sympatholytic receptors.

5-hydroxytryptamine (5-HT) modulates noradrenergic activity in different cardiovascular territories, but its effect on the mesenteric vasopressor outflow has not yet been clarified. This study investigated the in vivo serotonergic influence, characterizing 5-HT receptors implicated, in sympathetic innervation of mesenteric vasculature. Wistar rats were anaesthetised and prepared for the in situ autoperfused rat mesentery, monitoring systemic blood pressure (SBP), heart rate (HR) and mesenteric perfusion pressure (MPP). Electrical stimulation of mesenteric sympathetic nerves resulted in frequency-dependent increases in MPP (9 ± 1.6, 25.7 ± 3.9 and 60.2 ± 5 mmHg for 2, 4 and 8 Hz, respectively), without altering SBP or HR. 5-HT (1-25 µg/kg), 5-carboxamidotryptamine (5-HT1/7 agonist; 25 µg/kg) or L-694,247 (5-HT1D agonist; 1-25 µg/kg) i.a. bolus inhibited vasopressor responses by mesenteric nerves electrical stimulation, unlike i.a. bolus of agonists 8-OH-DPAT (5-HT1A ), CGS-12066B (5-HT1B ), BRL 54443 (5-HT1e/1F ), α-methyl-5-HT (5-HT2 ), 1-PBG (5-HT3 ), cisapride (5-HT4 ) or AS-19 (5-HT7 ) (25 µg/kg each). Interestingly, i.a. L-694,247 (25 µg/kg) also reduced the exogenous norepinephrine-induced vasoconstrictions. Pretreatment with selective 5-HT1D receptor antagonist, LY310762 (1 mg/kg, i.v.), completely abolished L-694,247- and 5-HT-induced mesenteric sympathoinhibition. Furthermore, ELISA analysis confirmed 5-HT1D receptors expression in mesenteric artery. These findings suggest that serotonergic mechanisms-induced sympathoinhibition of mesenteric noradrenergic outflow is mediated by pre and/or postjunctional 5-HT1D receptors.

Long-term estradiol exposure is a direct mitogen for insulin/EGF-primed endometrial cells and drives PTEN loss-induced hyperplasic growth.

Loss of tumor-suppressor PTEN is the most common alteration in endometrial carcinoma. However, the relationship between loss of PTEN, growth factors [eg, insulin/insulin-like growth factor (IGF)-1], epidermal growth factor (EGF), and hyperestrogenism in the development of endometrial carcinoma is still controversial. By using three-dimensional (3D) cultures of PTEN(+/+) and PTEN(+/-) endometrial epithelial cells, we investigated the effects of EGF, insulin/IGF, and estradiol in endometrial cell proliferation. We have previously demonstrated that 3D cultures of endometrial cells require EGF and insulin/IGF to proliferate. Herein, we demonstrate that, in the presence of EGF and insulin/IGF, long-term estradiol treatment directly induces proliferation of 3D cultures. Moreover, we show that the mitogenic effects of estradiol require the presence of insulin/IGF and EGF, because withdrawal of such factors completely abolishes estradiol-induced proliferation. In the presence of EGF and insulin/IGF, PTEN(+/-) and PTEN(+/+) spheroids display a similar rate of proliferation. However, the addition of estradiol causes an exaggerated proliferation of PTEN(+/-) cultures, leading to formation of complex structures, such as those observed in endometrial hyperplasia or carcinoma. In summary, we demonstrate that EGF and insulin/IGF prime endometrial epithelial cells to direct the mitogenic effects of estradiol. Furthermore, PTEN deficiency results in enhanced responsiveness to this combination, leading to the development of hyperplasia of endometrial cells in culture.

Chronic sarpogrelate treatment improves renal sympathetic hyperactivity in experimental diabetes.

Diabetes and derived complications, especially diabetic nephropathy and neuropathy annually cause great morbimortality worldwide. 5-hydroxytryptamine (5-HT) acts as a modulator of renal sympathetic input and vascular tone. In this line, 5-HT2 receptor blockade has been linked with reduced incidence and progression of diabetic microvascular alterations. In this work, we aimed to determine, in diabetic rats, whether 5-HT2 blockade ameliorates renal function and to characterize the serotonergic modulatory action on renal sympathetic neurotransmission. Diabetes was induced in male Wistar rats by alloxan administration (150 mg/kg, s.c.), and sarpogrelate (30 mg/kg·day, p.o.; 5-HT2 antagonist) was administered for 14 days (DM-S). Normoglycemic and diabetic (DM) animals were maintained as aged-matched controls. At 28th day, DM-S animals were anesthetized and prepared for the in situ autoperfusion of the kidney. Renal vasoconstrictor responses were induced electrically or by i.a. noradrenaline (NA) administration. The role of 5-HT and selective 5-HT agonist/antagonist were studied on these renal vasopressor responses. Sarpogrelate treatment decreased renal sympathetic-induced vasopressor responses, reduced renal hypertrophy and kidney damage markers increased in DM. Intraarterial 5-HT inhibited the sympathetic-induced renal vasoconstrictions, effect reproduced by 5-CT, AS-19, L-694,247 and LY 344864 (5-HT1/5/7, 5-HT7, 5-HT1D and 5-HT1F receptor agonists, respectively). Blocking 5-HT1D/1F/7 receptors completely abolished the 5-CT sympatho-inhibition. NA vasoconstrictions were not altered by any of the 5-HT agonists tested. Thus, in experimental diabetes, chronic sarpogrelate treatment reduces renal damage markers, kidney hypertrophy and renal sympathetic hyperactivity and modifies serotonergic modulation of renal sympathetic neurotransmission, causing a sympatho-inhibition by prejunctional 5-HT1D/1F and 5-HT7 activation.

KSR1 is overexpressed in endometrial carcinoma and regulates proliferation and TRAIL-induced apoptosis by modulating FLIP levels.

The Raf/MEK/extracellular signal-regulated kinase (ERK) pathway participates in many processes altered in development and progression of cancer in human beings such as proliferation, transformation, differentiation, and apoptosis. Kinase suppressor of Ras 1 (KSR1) can interact with various kinases of the Raf/MEK/extracellular signal-regulated kinase pathway to enhance its activation. The role of KSR1 in endometrial carcinogenesis was investigated. cDNA and tissue microarrays demonstrated that expression of KSR1 was up-regulated in endometrial carcinoma. Furthermore, inhibition of KSR1 expression by specific small hairpin RNA resulted in reduction of both proliferation and anchorage-independent cell growth properties of endometrial cancer cells. Because inhibition of apoptosis has a pivotal role in endometrial carcinogenesis, the effects of KSR1 in regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis were investigated. KSR1 knock-down sensitized resistant endometrial cell lines to both TRAIL- and Fas-induced apoptosis. Sensitization to TRAIL and agonistic anti-Fas antibody was caused by down-regulation of FLIP (FLICE-inhibitory protein). Also investigated was the molecular mechanism by which KSR1 regulates FLIP protein levels. It was demonstrated that KSR1 small hairpin RNA did not affect FLIP transcription or degradation. Rather, FLIP down-regulation was caused by Fas-associated death domain protein-dependent inhibition of FLIP translation triggered after TRAIL stimulation in KSR1-silenced cells. Re-expression of heterologous KSR1 in cells with down-regulated endogenous KSR1 restored FLIP protein levels and TRAIL resistance. In conclusion, KSR1 regulates endometrial sensitivity to TRAIL by regulating FLIP levels.

Assessing dynamic change in muscle during treatment of patients with cancer: Precision testing standards.

BACKGROUND: Computed tomography images acquired during routine cancer care provide an opportunity to determine body composition with accuracy and precision. Quantification of skeletal muscle is of interest owing to its association with clinical outcomes. However, the standards of precision testing considered mandatory in other areas of radiology are lacking from the literature in this area. We aim to describe the change in skeletal muscle over time at different anatomical levels using the precision error. METHODS: Thirty-eight male patients with squamous cell carcinoma of the head and neck were evaluated at two time points encompassing their treatment plan. Precision testing consisted of analyzing the cross-sectional area (CSA) of the skeletal muscle and total adipose tissue of 76 CT studies (38 images at baseline repeated twice and 38 follow-up images repeated twice) measured by a skilled observer. The % coefficient of variation (%CV), the root-mean-square standard deviation (RMS SD) and the corresponding 95% least significant change (LSC) were calculated for four anatomical levels: upper arm, thigh, chest and abdomen. RESULTS: The median time between scans was 223.6 (SD 31.2) days. Precision error (% CV) for total skeletal muscle cross sectional area was 0.86% for upper arm, 0.26% for thigh, 0.39% for chest and 0.63% for abdomen. The corresponding LSC values in upper arm, thigh, chest and abdomen were 2.4%, 0.7%, 1.1% and 1.8%, respectively. Based on the LSC for RMS SD, patients were classified in two categories according to muscle cross-sectional area: stable (i.e within LSC value) or gained and loss. To compare the four anatomical levels, the proportion of patients with muscle loss exceeding the LSC value was 74.3% for arm, 86.2% for thigh, 82.9% for chest and 76.3% for abdomen. For these same anatomic regions, the mean muscle loss for those patients classified below the LSC was 14.6% (SD 9.3), 13.4% (SD 7.8), 11.9% (SD 6.5) and 11.6% (SD 5.5), respectively. Only the loss of muscle area was significantly higher in thigh (p = 0.023), using L3 as the reference level. CONCLUSIONS: We recommend the uniform use of a standard precision test when reporting muscle change over time. LSC values vary from 0.7 to 2.4% depending on anatomic site; with the lowest precision error to detect change in the thigh. Based on this analysis, muscle wasting appears to be systemic and while present in limbs and trunk is significantly higher in the thigh than in the chest, abdomen or upper arm.

Effect of sarpogrelate treatment on 5-HT modulation of vascular sympathetic innervation and platelet activity in diabetic rats.

This study aimed to investigate whether the 5-HT2 receptor blockade alters the 5-HT effect on vascular sympathetic neurotransmission and platelet activation in type 1 diabetes. 28-day diabetes was obtained by alloxan (150 mg/kg; s.c.) in male Wistar rats, administering sarpogrelate (5-HT2 blocker; 30 mg/kg/day; p.o.) for 14 days. Blood glucose and body weight were monitored for 28 days. After 4 weeks of diabetes induction, food and drink intake, urine, plasma-platelet 5-HT, and platelet activation were determined in normoglycemic, non-treated diabetic and sarpogrelate-treated diabetic rats. Another set of diabetic rats were pithed to run the vascular sympathetic stimulation or exogenous noradrenaline administration, examining the induced vasoconstrictor responses. Sarpogrelate treatment significantly reduced drink intake and urine, whereas BW gain, hyperglycemia, and food intake were not modified in diabetic rats. The platelet activation and plasma 5-HT concentration were decreased (increasing the stored 5-HT platelet) by 5-HT2 blockade in diabetic animals. The sympathetic-induced vasoconstrictions were higher in non-treated than in sarpogrelate-treated diabetic rats. 5-HT inhibited these vasopressor responses, reproduced exclusively by the 5-HT1/5/7 receptor agonist, 5-CT. The 5-CT-produced inhibition was partly reversed by 5-HT1D or 5-HT7 antagonists (LY310762 or SB-258719, respectively), and totally annulled by the mixture of LY310762+SB-258719. Noradrenaline-caused vasoconstrictions were also decreased by 5-CT. In conclusion, our results reveal that 14-day sarpogrelate treatment improves polydipsia and polyuria, reduces platelet hyperactivation, plasma 5-HT and the vascular sympathetic tone, and changes 5-HT receptors inhibiting noradrenergic drive in diabetic rats: pre and/or postjunctional 5-HT1D/7 are involved in the sympatho-inhibition.

Oral fluoxetine treatment changes serotonergic sympatho-regulation in experimental type 1 diabetes.

AIMS: This study investigated whether fluoxetine treatment changes the 5-HT regulation on vascular sympathetic neurotransmission in type 1 diabetes. MAIN METHODS: Four-week diabetes was obtained by a single alloxan s.c. administration in male Wistar rats, administering fluoxetine for 14 days (10 mg/kg/day; p.o.). Systolic blood pressure, heart rate, glycaemia, body weight (BW) evolution, creatinine, and blood urea nitrogen (BUN) were monitored. Afterward, rats were pithed to perform the vascular sympathetic stimulation. 5-HT1A/1D/2A receptors expression was analysed by Western blot in thoracic aorta. Both i.v. norepinephrine and the electrical stimulation of the spinal sympathetic drive evoked vasoconstrictor responses. KEY FINDINGS: Fluoxetine treatment significantly reduced the BW gain, hyperglycaemia, creatinine, and BUN in diabetic rats. The electrical-produced vasopressor responses were greater in untreated than in fluoxetine-treated diabetic rats. 5-HT decreased the sympathetic-produced vasopressor responses. While 5-CT, 8-OH-DPAT and L-694,247 (5-HT1/7, 5-HT1A and 5-HT1D agonists, respectively) reproduced 5-HT-evoked inhibition, the 5-HT2 activation by α-methyl-5-HT augmented the vasoconstrictions. The 5-CT sympatho-inhibition was reversed by 5-HT1A plus 5-HT1D antagonists (WAY-100,635 and LY310762, respectively), whereas ritanserin (5-HT2A antagonist) blocked the α-methyl-5-HT potentiating effect. Norepinephrine-generated vasoconstrictions were increased or diminished by α-methyl-5-HT or 5-CT, respectively. 5-HT1A/1D/2A receptors were expressed at vascular level, being 5-HT1A expression increased by fluoxetine in diabetic rats. SIGNIFICANCE: Our findings suggest that fluoxetine improves metabolic and renal profiles, changes the vasopressor responses, and 5-HT receptors modulating sympathetic activity in diabetic rats: 5-HT1A/1D are involved in the sympatho-inhibition, while 5-HT2A is implicated in the sympatho-potentiation, being both effects pre and/or postjunctional in nature.

Nuclear factor-κB2/p100 promotes endometrial carcinoma cell survival under hypoxia in a HIF-1α independent manner.

Endometrial carcinoma (EC) is a common female cancer, treated mainly by surgery and adjuvant radiotherapy. Relapse following treatment is associated with increased risk of metastases. Hypoxia, a common microenvironment in solid tumors, correlates with malignant progression, rendering tumors resistant to ionizing therapy. Hence, we assessed here the immunohistochemical expression of hypoxia-inducible factor-1α (HIF-1α) and members of the NF-κB family in 82 primary EC and 10 post-radiation recurrences of EC. Post-radiation recurrences were highly hypoxic, with a higher expression of HIF-1α and also RelA (p65) and p52 when compared with primary EC. We next investigated the effects of hypoxia on EC cell lines. We found that EC cell lines are highly resistant to hypoxia-induced apoptosis. We thus focused on the molecular mechanisms involved in conferring hypoxic cell death resistance. We show that in addition to the classical NF-κB, hypoxia activates the alternative NF-κB pathway. To characterize the upstream kinases involved in the activation of these pathways, we used lentiviral-mediated knockdown and mouse embryonic fibroblasts lacking IKKα and IKKß kinases. Both IKKα and IKKß kinases are required for RelA (p65) and p100 accumulation, whereas p52 processing under hypoxia is IKKα dependent. Furthermore, Ishikawa endometrial cell line harboring either RelA (p65) or p52 short-hairpin RNA was sensitive to hypoxia-induced cell death, indicating that, in addition to the known prosurvival role of RelA (p65) under hypoxia, alternative NF-κB pathway also enhances hypoxic survival of EC cells. Interestingly, although HIF-1α controlled classical NF-κB activation pathway and survival under hypoxia through RelA (p65) nuclear accumulation, the alternative pathway was HIF-1α independent. These findings have important clinical implications for the improvement of EC prognosis before radiotherapy.

A novel three-dimensional culture system of polarized epithelial cells to study endometrial carcinogenesis.

Development of three-dimensional (3D) cultures that mimic in vivo tissue organization has a pivotal role in the investigation of the involvement of cell adhesion and polarity genes in the pathogenesis of epithelial cancers. Here we describe a novel 3D culture model with primary mouse endometrial epithelial cells. In this model, isolated endometrial epithelial cells develop single-lumened, polarized glandular structures resembling those observed in endometrial tissue. Our in vitro 3D culture model of endometrial glands requires the use of serum-free defined medium with only epidermal growth factor and insulin as growth supplements and 3% Matrigel as reconstituted extracellular matrix. Under these culture conditions, glands of epithelial cells displaying typical apicobasal polarity and proper positioning of tight and adherent junctions are formed by hollowing as early as 7 to 8 days in culture. Addition of the phosphatidylinositol 3-kinase inhibitor LY294002 completely inhibits bromodeoxyuridine incorporation and cyclinD1 expression, confirming that in vitro growth of endometrial glands depends on phosphatidylinositol 3-kinase/Akt signaling. To prove that our culture method is a good model to study endometrial carcinogenesis, we knocked down E-cadherin or phosphatase and tensin homolog expression by lentivirus-delivered short hairpin RNAs. Down-regulation of E-cadherin resulted in complete loss of epithelial cell polarity and glandular formation, whereas phosphatase and tensin homolog down-regulation resulted in increased proliferation of glandular epithelial cells. These properties indicate that our 3D culture model is suitable to study the effect of growth factors, drugs, and gene alterations in endometrial carcinogenesis and to study normal endometrial biology/physiology.

Biologic treatments for psoriasis: the cost of alternative methods of dispensation of medication in Spain.

OBJECTIVE: Hospital pharmacy services have adapted to the COVID­19 pandemic. The aim of the study is to determine the economic  consequences of replacing hospital pharmacy dispensation with other  dispensing methods in the context of biological treatments for psoriasis in  Spain. METHOD: Multiple dispensation scenarios were evaluated, combining different  dispensation frequencies and sites, and telepharmacy followup intervals. Self- injectable biological medicines for psoriasis (interleukin and tumour necrosis  factor alpha inhibitors) were included. All costs (in 2020 euros) were  considered from the perspective of the National Health System. RESULTS: The annual cost of hospital pharmacy-based dispensations every 4  weeks combined with telepharmacy monitoring at each administration ranged  from €194.9 to €2,088.0 per patient. Across the different simulated scenarios,  biological medicines associated with the lowest cost were those administered  less frequently (every 12 weeks). CONCLUSIONS: In the post-COVID-19 era, new models of hospital  pharmaceutical care that include changes in drug dispensation and  telepharmacy strategies will have economic consequences for the National  Health System that merit consideration.

Objetivo: Los servicios de farmacia hospitalaria se han adaptado a la pandemia de COVID-19. El objetivo del estudio es determinar las  consecuencias económicas de sustituir la dispensación de medicamentos en el servicio de farmacia hospitalaria por otros métodos de dispensación en el  contexto de los tratamientos biológicos para la psoriasis en España.Método: Se evaluaron múltiples escenarios de dispensación, combinando diferentes frecuencias y lugares de dispensación, y frecuencias del seguimiento de telefarmacia. Se incluyeron los medicamentos biológicos autoinyectables para la psoriasis (inhibidores de interleucinas y del  factor de necrosis tumoral alfa). Todos los costes (euros de 2020) se  consideraron desde la perspectiva del Sistema Nacional de Salud.Resultados: Considerando la dispensación en la farmacia hospitalaria, la  frecuencia de dispensación cada 4 semanas y la telefarmacia en cada  administración, el coste anual de dispensación por paciente osciló entre 194,9  € y 2.088,0 €. En los diferentes escenarios simulados, los fármacos biológicos  que se asociaron a un coste inferior fueron los que se administran de forma  más espaciada en el tiempo (cada 12 semanas).Conclusiones: En la era post-COVID-19, los nuevos modelos de atención farmacéutica hospitalaria que consideran cambios en la dispensación  farmacológica y la telefarmacia tendrán consecuencias económicas para el Sistema Nacional de Salud que merecen consideración.

Clinical outcomes after more conservative management of patent ductus arteriosus in preterm infants.

OBJECTIVE: Management of patent ductus arteriosus is still controversial. This study aimed to describe the impact of a more conservative approach on treatment rates and on main outcomes of prematurity, especially in preterm infants with <26 weeks of gestation. METHOD: Clinical charts review of infants ≤30 weeks with patent ductus arteriosus between 2009 and 2016 at two centers. In 2011, the authors changed patent ductus arteriosus management: in first period (2009-2011), patients who failed medical treatment underwent surgical closure; in second period (2012-2016), only those with cardiopulmonary compromise underwent surgical ligation. Medical treatment, surgical closure, mortality, and survival-without-morbidity were compared. RESULTS: This study included 188 patients (27±2 weeks, 973±272 grams); 63 in P1 and 125 in P2. In P2, significantly lower rates of medical treatment (85.7% P1 versus 56% P2, p<0.001) and surgical closure (34.5% P1 versus 16.1% P2, p<0.001) were observed. No differences were found in chronic lung disease (28.8% versus 13.9%, p=0.056), severe retinopathy of prematurity (7.5% versus 11.8%, p=0.403), necrotizing enterocolitis (15.5% versus 6.9%, p=0.071), severe intraventricular hemorrhage (25.4% versus 18.4%, p=0.264), mortality (17.5% versus 15.2%, p=0.690) or survival-without-morbidity adjusted OR=1.10 (95% CI: 0.55-2.22); p=0.783. In P2, 24.5% patients were discharged with patent ductus arteriosus. The subgroup born between 23 and 26 weeks (n=82) showed significant differences: lower incidence of chronic lung disease (50% versus 19.6%, p=0.019) and more survival-without-morbidity (20% versus 45.6%, p=0.028) were found. CONCLUSION: A conservative approach in preterm infants with patent ductus arteriosus can avoid medical and surgical treatments, without a significant impact in survival-without-morbidity. However, two-thirds of preterm infants under 26 weeks are still treated.

Role of peripheral 5-HT5A receptors in 5-HT-induced cardiac sympatho-inhibition in type 1 diabetic rats.

5-HT inhibits cardiac sympathetic neurotransmission in normoglycaemic rats, via 5-HT1B, 5-HT1D and 5-HT5A receptor activation. Since type 1 diabetes impairs the cardiac sympathetic innervation leading to cardiopathies, this study aimed to investigate whether the serotonergic influence on cardiac noradrenergic control is altered in type 1 diabetic rats. Diabetes was induced in male Wistar rats by streptozotocin (50 mg/kg, i.p.). Four weeks later, the rats were anaesthetized, pithed and prepared for producing tachycardic responses by electrical preganglionic stimulation (C7-T1) of the cardioaccelerator sympathetic outflow or i.v. noradrenaline bolus injections. Immunohistochemistry was performed to study 5-HT1B, 5-HT1D and 5-HT5A receptor expression in the stellate ganglion from normoglycaemic and diabetic rats. In the diabetic group, i) i.v. continuous infusions of 5-HT induced a cardiac sympatho-inhibition that was mimicked by the 5-HT1/5A agonist 5-carboxamidotryptamine (without modifying noradrenaline-induced tachycardia), but not by the agonists indorenate (5-HT1A), CP 93,129 (5-HT1B), PNU 142633 (5-HT1D), or LY344864 (5-HT1F); ii) SB 699551 (5-HT5A antagonist; i.v.) completely reversed 5-CT-induced cardiac sympatho-inhibition; and iii) 5-HT5A receptors were more expressed in the stellate ganglion compared to normoglycaemic rats. These results show the prominent role of the peripheral 5-HT5A receptors prejunctionally inhibiting the cardiac sympathetic drive in type 1 diabetic rats.

Hypertension exhibits 5-HT4 receptor as a modulator of sympathetic neurotransmission in the rat mesenteric vasculature.

Sympathetic overdrive is a key player in hypertension, where the mesenteric vasculature plays a relevant role in modulating blood pressure. Although 5-HT inhibits noradrenergic mesenteric neurotransmission in normotensive rats, its effect on the mesenteric sympathetic drive in hypertensive rats has not been studied. We investigated the influence of in vivo 5-HT by characterizing the implicated serotonergic receptors on the mesenteric sympathetic outflow in rats with N-nitro-L-arginine methyl ester (L-NAME)-induced hypertension. Hypertension was induced in male Wistar rats by L-NAME administration (30 mg/kg per day; 21 days) in drinking water. The rats were anesthetized (sodium pentobarbital; 60 mg/kg, i.p.), prepared for the in situ autoperfused rat mesentery, and subjected for monitoring their systemic blood pressure (SBP), heart rate (HR), and mesenteric perfusion pressure (MPP). Electrical stimulation of mesenteric sympathetic nerves resulted in frequency-dependent increases in MPP without altering SBP or HR. The 5-HT and cisapride (5-HT4 agonist) i.a. bolus (1-25 µg/kg) inhibited vasopressor responses by electrical stimulation of the mesenteric nerves, unlike an i.a. bolus (25 µg/kg each) of the agonist 5-carboxamidotryptamine (5-HT1/7 agonist), α-methyl-5-HT (5-HT2), or 1-PBG (5-HT3). However, i.a. cisapride (25 µg/kg) did not affect the noradrenaline-induced vasoconstriction in the mesenteric vasculature. Administration of the selective 5-HT4 receptor antagonist GR 125487 (1 mg/kg, i.v.) completely abolished cisapride- and 5-HT-evoked mesenteric sympatholytic effects. Additionally, ELISA analysis demonstrated higher 5-HT4 receptor expression in mesenteric arteries from L-NAME-hypertensive compared with normotensive rats. Our findings suggest that L-NAME-induced hypertension modifies the 5-HT modulation of the rat mesenteric sympathetic drive: prejunctional 5-HT4 receptors are involved in the serotonergic sympathoinhibitory effect.