Evidence for the involvement of TRPV2 channels in the modulation of vascular tone in the mouse aorta.

AIMS: Transient receptor potential vanilloid 2 (TRPV2) channels are expressed in both smooth muscle and endothelial cells and participate in vascular mechanotransduction and sensing of high temperatures and lipids. Nevertheless, the impact of TRPV2 channel activation by agonists on the coordinated and cell-type specific modulation of vasoreactivity is unknown. MAIN METHODS: Aorta from 2- to 4-months-old male Oncins France 1 mice was dissected and mounted in tissue baths for isometric tension measurements. TRPV2 channel expression was assessed by immunofluorescence and western blot in mice aortas and in cultured A7r5 rat aortic smooth muscle cells. KEY FINDINGS: TRPV2 channels were expressed in all three mouse aorta layers. Activation of TRPV2 channels with probenecid evoked endothelium-dependent relaxations through a mechanism that involved activation of smooth muscle Kir and Kv channels. In addition, TRPV2 channel inhibition with tranilast increased endothelium-independent relaxations to probenecid and this effect was abrogated by the KATP channel blocker glibenclamide, revealing that smooth muscle TRPV2 channels induce negative feedback on probenecid relaxations mediated via KATP channel inhibition. Exposure to the NO donor sodium nitroprusside increased TRPV2 channel translocation to the plasma membrane in cultured smooth muscle cells and enhanced negative feedback on probenecid relaxations. SIGNIFICANCE: In conclusion, we present the first evidence that TRPV2 channels may modulate vascular tone through a balance of opposed inputs from the endothelium and the smooth muscle leading to net vasodilation. The fact that TRPV2 channel-induced activity can be amplified by NO emphasizes the pathophysiological relevance of these findings.

An Imidazoline 2 Receptor Ligand Relaxes Mouse Aorta via Off-Target Mechanisms Resistant to Aging.

Imidazoline receptors (IR) are classified into three receptor subtypes (I1R, I2R, and I3R) and previous studies showed that regulation of I2R signaling has neuroprotective potential. In order to know if I2R has a role in modulating vascular tone in health and disease, we evaluated the putative vasoactive effects of two recently synthesized I2R ligands, diethyl (1RS,3aSR,6aSR)-5-(3-chloro-4-fluorophenyl)-4,6-dioxo-1-phenyl-1,3a,4,5,6,6a-hexahydropyrrolo[3,4-c]pyrrole -1-phosphonate (B06) and diethyl [(1-(3-chloro-4-fluorobenzyl)-5,5-dimethyl-4-phenyl-4,5-dihydro-1H-imidazol-4-yl]phosphonate] (MCR5). Thoracic aortas from Oncins France 1 (3- to 4-months-old) and C57BL/6 (3- to 4- and 16- to 17-months-old mice) were mounted in tissue baths to measure isometric tension. In young mice of both strains, MCR5 induced greater relaxations than either B06 or the high-affinity I2R selective ligand 2-(2-benzofuranyl)-2-imidazoline (2-BFI), which evoked marginal responses. MCR5 relaxations were independent of I2R, as IR ligands did not significantly affect them, involved activation of smooth muscle KATP channels and inhibition of L-type voltage-gated Ca2+ channels, and were only slightly modulated by endothelium-derived nitric oxide (negatively) and prostacyclin (positively). Notably, despite the presence of endothelial dysfunction in old mice, MCR5 relaxations were preserved. In conclusion, the present study provides evidence against a functional contribution of I2R in the modulation of vascular tone in the mouse aorta. Moreover, the I2R ligand MCR5 is an endothelium-independent vasodilator that acts largely via I2R-independent pathways and is resistant to aging. We propose MCR5 as a candidate drug for the management of vascular disease in the elderly.

Role of serendipity in the discovery of classical antidepressant drugs: Applying operational criteria and patterns of discovery.

The role played by serendipity in the origin of modern psychopharmacology has proven to be controversial in scientific literature. In its original meaning (Walpole), serendipity refers to discoveries made through a combination of accidents and sagacity. We have implemented an operational definition of serendipity based on finding something unexpected or unintended, regardless of the systematic process that led to the accidental observation, and we have established four different patterns of serendipitous attributability. In this paper, we have analyzed the role of serendipity in the discovery and development of classical antidepressant drugs, tricyclic antidepressants and monoamine oxidase inhibitors as well as heterocyclic, "atypical" or "second generation" antidepressants. The discovery of the antidepressant properties of imipramine and iproniazid, the prototypes of tricyclic antidepressants and monoamine oxidase inhibitors, respectively, fits the mixed type II pattern; initial serendipitous discoveries (imipramine was an antipsychotic and iproniazid was an anti-tuberculosis agent) led secondarily to non-serendipitous discoveries. But the other components of these two families of drugs were developed specifically as antidepressants, modifying the chemical structure of the series leaders, thereby allowing all of them to be included in the type IV pattern, characterized by the complete absence of serendipity. Among the heterocyclic drugs, mianserin (originally developed as an antihistamine) also falls into the type II pattern.

MicroRNAs and Oxidative Stress: An Intriguing Crosstalk to Be Exploited in the Management of Type 2 Diabetes.

Type 2 diabetes is a chronic disease widespread throughout the world, with significant human, social, and economic costs. Its multifactorial etiology leads to persistent hyperglycemia, impaired carbohydrate and fat metabolism, chronic inflammation, and defects in insulin secretion or insulin action, or both. Emerging evidence reveals that oxidative stress has a critical role in the development of type 2 diabetes. Overproduction of reactive oxygen species can promote an imbalance between the production and neutralization of antioxidant defence systems, thus favoring lipid accumulation, cellular stress, and the activation of cytosolic signaling pathways, and inducing ß-cell dysfunction, insulin resistance, and tissue inflammation. Over the last few years, microRNAs (miRNAs) have attracted growing attention as important mediators of diverse aspects of oxidative stress. These small endogenous non-coding RNAs of 19-24 nucleotides act as negative regulators of gene expression, including the modulation of redox signaling pathways. The present review aims to provide an overview of the current knowledge concerning the molecular crosstalk that takes place between oxidative stress and microRNAs in the physiopathology of type 2 diabetes, with a special emphasis on its potential as a therapeutic target.

NT3/TrkC Pathway Modulates the Expression of UCP-1 and Adipocyte Size in Human and Rodent Adipose Tissue.

Neurotrophin-3 (NT3), through activation of its tropomyosin-related kinase receptor C (TrkC), modulates neuronal survival and neural stem cell differentiation. It is widely distributed in peripheral tissues (especially vessels and pancreas) and this ubiquitous pattern suggests a role for NT3, outside the nervous system and related to metabolic functions. The presence of the NT3/TrkC pathway in the adipose tissue (AT) has never been investigated. Present work studies in human and murine adipose tissue (AT) the presence of elements of the NT3/TrkC pathway and its role on lipolysis and adipocyte differentiation. qRT-PCR and immunoblot indicate that NT3 (encoded by NTF3) was present in human retroperitoneal AT and decreases with age. NT3 was also present in rat isolated adipocytes and retroperitoneal, interscapular, perivascular, and perirenal AT. Histological analysis evidences that NT3 was mainly present in vessels irrigating AT close associated to sympathetic fibers. Similar mRNA levels of TrkC (encoded by NTRK3) and ß-adrenoceptors were found in all ATs assayed and in isolated adipocytes. NT3, through TrkC activation, exert a mild effect in lipolysis. Addition of NT3 during the differentiation process of human pre-adipocytes resulted in smaller adipocytes and increased uncoupling protein-1 (UCP-1) without changes in ß-adrenoceptors. Similarly, transgenic mice with reduced expression of NT3 (Ntf3 knock-in lacZ reporter mice) or lacking endothelial NT3 expression (Ntf3flox1/flox2;Tie2-Cre+/0) displayed enlarged white and brown adipocytes and lower UCP-1 expression. Conclusions: NT3, mainly released by blood vessels, activates TrkC and regulates adipocyte differentiation and browning. Disruption of NT3/TrkC signaling conducts to hypertrophied white and brown adipocytes with reduced expression of the thermogenesis marker UCP-1.

Stenosis coexists with compromised α1-adrenergic contractions in the ascending aorta of a mouse model of Williams-Beuren syndrome.

Williams-Beuren syndrome (WBS) is a rare disorder caused by a heterozygous deletion of 26-28 contiguous genes that affects the brain and cardiovascular system. Here, we investigated whether WBS affects aortic structure and function in the complete deletion (CD) mouse model harbouring the most common deletion found in WBS patients. Thoracic aortas from 3-4 months-old male CD mice and wild-type littermates were mounted in wire myographs or were processed for histomorphometrical analysis. Nitric oxide synthase (NOS) isoforms and oxidative stress levels were assessed. Ascending aortas from young adult CD mice showed moderate (50%) luminal stenosis, whereas endothelial function and oxidative stress were comparable to wild-type. CD mice showed greater contractions to KCl. However, α1-adrenergic contractions to phenylephrine, but not with a thromboxane analogue, were compromised. Decreased phenylephrine responses were not affected by selective inducible NOS blockade with 1400 W, but were prevented by the non-selective NOS inhibitor L-NAME and the selective neuronal NOS inhibitor SMTC. Consistently, CD mice showed increased neuronal NOS expression in aortas. Overall, aortic stenosis in CD mice coexists with excessive nNOS-derived NO signaling that compromises ascending aorta α1-adrenergic contractions. We suggest that increased neuronal NOS signaling may act as a physiological 'brake' against the detrimental effects of stenosis.

Bloqueadores beta: perspectiva histórica y mecanismos de acción / Beta-blockers: historical perspective and mechanisms of action

Los bloqueadores beta son moléculas ampliamente utilizadas y capaces de antagonizar los receptores adrenérgicos (RA) beta, pertenecen a la familia de receptores acoplados a proteínas G y reciben el estímulo de las catecolaminas endógenas. Tras su estimulación, se activan cascadas intracelulares que en última instancia originan la contracción cardiaca o la dilatación vascular, según el subtipo y su ubicación. Se han descrito 3 subtipos, que se expresan de manera diferenciada en el organismo (RA-ß1, ß2 y ß3), y el subtipo ß1 es el más abundante en el corazón. Desde su descubrimiento, los RA-ß se han convertido en diana para combatir las enfermedades cardiovasculares. Desde su invención por James Black a finales de los años cincuenta, los bloqueadores beta han supuesto una revolución en la terapia cardiovascular. Hasta ahora se dispone de 3 generaciones: los bloqueadores beta no selectivos, los bloqueadores beta cardioselectivos (antagonista selectivo de ß1) y los bloqueadores beta vasodilatadores. Estos constituyen la tercera generación y son capaces de bloquear los ß1 además de tener actividad vasodilatadora, bien bloqueando los RA-alfa1 o activando los RA-ß3. Los bloqueadores beta todavía se utilizan ampliamente en la clínica tras más de 50 años desde la introducción del propranolol en el mercado por su capacidad para reducir la frecuencia cardiaca y, por lo tanto, la demanda miocárdica de oxígeno en el caso de una angina

Beta-blockers are widely used molecules that are able to antagonize ß-adrenergic receptors (ARs), which belong to the G protein-coupled receptor family and receive their stimulus from endogenous catecholamines. Upon ß-AR stimulation, numerous intracellular cascades are activated, ultimately leading to cardiac contraction or vascular dilation, depending on the relevant subtype and their location. Three subtypes have been described that are differentially expressed in the body (ß1-, ß2- and ß3-ARs), ß1 being the most abundant subtype in the heart. Since their discovery, ß-ARs have become an important target to fight cardiovascular disease. In fact, since their discovery by James Black in the late 1950s, ß-blockers have revolutionized the field of cardiovascular therapies. To date, 3 generations of drugs have been released: nonselective ß-blockers, cardioselective ß-blockers (selective ß1-antagonists), and a third generation of these drugs able to block ß1 together with extra vasodilation activity (also called vasodilating ß-blockers) either by blocking alfa1- or by activating ß3-AR. More than 50 years after propranolol was introduced to the market due to its ability to reduce heart rate and consequently myocardial oxygen demand in the event of an angina attack, ß-blockers are still widely used in clinics

Beta-blockers: Historical Perspective and Mechanisms of Action.

Beta-blockers are widely used molecules that are able to antagonize ß-adrenergic receptors (ARs), which belong to the G protein-coupled receptor family and receive their stimulus from endogenous catecholamines. Upon ß-AR stimulation, numerous intracellular cascades are activated, ultimately leading to cardiac contraction or vascular dilation, depending on the relevant subtype and their location. Three subtypes have been described that are differentially expressed in the body (ß1-, ß2- and ß3-ARs), ß1 being the most abundant subtype in the heart. Since their discovery, ß-ARs have become an important target to fight cardiovascular disease. In fact, since their discovery by James Black in the late 1950s, ß-blockers have revolutionized the field of cardiovascular therapies. To date, 3 generations of drugs have been released: nonselective ß-blockers, cardioselective ß-blockers (selective ß1-antagonists), and a third generation of these drugs able to block ß1 together with extra vasodilation activity (also called vasodilating ß-blockers) either by blocking α1- or by activating ß3-AR. More than 50 years after propranolol was introduced to the market due to its ability to reduce heart rate and consequently myocardial oxygen demand in the event of an angina attack, ß-blockers are still widely used in clinics.

Uric acid treatment after stroke modulates the Krüppel-like factor 2-VEGF-A axis to protect brain endothelial cell functions: Impact of hypertension.

Uric acid (UA) is a promising protective treatment in ischaemic stroke, but the precise molecular targets underlying its in vivo beneficial actions remain unclear. High concentrations of UA inhibit angiogenesis of cultured endothelial cells via Krüppel-like factor 2 (KLF)-induced downregulation of vascular endothelial growth factor (VEGF), a pro-angiogenic mediator that is able to increase blood-brain barrier (BBB) permeability in acute stroke. Here, we investigated whether UA treatment after ischaemic stroke protects brain endothelial cell functions and modulates the KLF2-VEGF-A axis. Transient intraluminal middle cerebral artery (MCA) occlusion/reperfusion was induced in adult male spontaneously hypertensive (SHR) rats and corresponding normotensive Wistar-Kyoto (WKY) rats. Animals received UA (16 mg/kg) or vehicle (Locke's buffer) i.v. at reperfusion. BBB permeability was evaluated by Evans blue extravasation to the brain and in human cerebral endothelial hCMEC/D3 cells under oxygen-glucose deprivation/re-oxygenation. Circulating VEGF-A levels were measured in rats and acute ischaemic stroke patients from the URICO-ICTUS trial. Angiogenesis progression was assessed in Matrigel-cultured MCA. Worse post-stroke brain damage in SHR than WKY rats was associated with higher hyperaemia at reperfusion, increased Evans blue extravasation, exacerbated MCA angiogenic sprouting, and higher VEGF-A levels. UA treatment reduced infarct volume and Evans blue leakage in both rat strains, improved endothelial cell barrier integrity and KLF2 expression, and lowered VEGF-A levels in SHR rats. Hypertensive stroke patients treated with UA showed lower levels of VEGF-A than patients receiving vehicle. Consistently, UA prevented the enhanced MCA angiogenesis in SHR rats by a mechanism involving KLF2 activation. We conclude that UA treatment after ischaemic stroke upregulates KLF2, reduces VEGF-A signalling, and attenuates brain endothelial cell dysfunctions leading to neuroprotection.

ß2-Adrenoceptors and GRK2 as Potential Biomarkers in Patients With Chronic Pulmonary Regurgitation.

Pulmonary regurgitation (PR) is a frequent complication after repair of congenital heart disease. Three different GRK isoforms (GRK2, GRK5, and GRK3) and two ß-adrenoceptors (ß1-AR and ß2-AR) are present in peripheral blood mononuclear cells (PBMC) and their expression changes as a consequence of the hemodynamic and neurohumoral alterations that occur in some cardiovascular diseases. Therefore, they could be useful as biomarkers in PR. A prospective study was conducted to describe the expression (TaqMan Gene Expression Assays) of ß-ARs and GRKs in PBMC isolated (Ficoll® gradient) from patients with severe PR before and after pulmonary valve replacement and establish if this expression correlates to clinical status. 23 patients with severe PR were included and compared with 22 healthy volunteers (controls). PR patients before the PVR had a significantly lower expression of ß2-AR (513.8 ± 261.2 mRNA copies) vs. controls (812.5 ± 497.2 mRNA copies), so as GRK2 expression (503.4 ± 364.9 copies vs. 858.1 ± 380.3 mRNA copies). The expression of ß2-AR and GRK2 significantly decreases in symptomatic and asymptomatic patients, as well as in patients under treatment with beta-blockers and non-treated patients. The expression of ß2-AR and GRK2 in PR patients recovers the normal values after pulmonary valve replacement (754,8 ± 77,1 and 897,8 ± 87,4 copies, respectively). Therefore, changes in the expression of ß2-AR and GRK2 in PBMC of PR patients, could be considered as potential biomarkers to determine clinical decisions.

Dexamethasone Preconditioning in Cardiac Procedures Reduces Decreased Antithrombin Activity and Is Associated to Beneficial Outcomes: Role of Endothelium.

Introduction: Decreased antithrombin (AT) activity in patients scheduled for cardiovascular surgery under cardiopulmonary bypass (CPB) is related to increased postoperative complications and hospitalization time. Indirect evidence suggests that glucocorticoids mitigate this decreased AT activity. To better understand the beneficial effects of AT we have analyzed: (i) the clinical relevance of acute dexamethasone (DX) administration before cardiac surgery on AT activity, (ii) the modulation by DX of AT expression in human endothelial cells (hECs), (iii) the activity of AT on migration and angiogenesis of hECs, or on angiogenesis of rat aorta. Methods: A retrospective cohort study in patients undergoing aortic valve replacement surgery was designed to evaluate the effect of DX administration on AT activity at five separate time points: preoperatively, during CPB, at intensive care unit admission and at 12 and 24 h post-intervention. We have analyzed also clinical differences in postoperative outcomes as safety and the length of stay in hospitalization. Changes in mRNA levels of AT induced by DX were determined by qRT-PCR in human coronary (hCEC), aorta (hAEC) and cardiac microvasculature (hCMEC) endothelial cells. AT activity on migration and angiogenesis were also assayed. Angiogenic growth of rat aortic rings incubated in Matrigel® was determined in presence and absence of AT. Results: The cohort comprised 51 patients in the control group and 29 patients in the group receiving dexamethasone. Preoperative DX supplementation reduced intraoperative decrease of AT activity (67.71 ± 10.49% DX treated vs. 58.12 ± 9.11% untreated, p < 0.001) that could be related to a decrease in the hospitalization time (7.59 ± 4.08 days DX treated vs. 13.59 ± 16.00 days untreated, p = 0.014). Treatment of hECs with 500 nM DX slightly increased AT expression. Incubation with 0.5 and 1 IU/mL of AT increased migration and angiogenesis in hCAECs and hAECs, but not in hCMECs. The same concentrations of AT potentiated angiogenic sprouting of new vessels from rat aorta. Conclusion: Preoperative DX supplementation could be an interesting procedure to avoid excessive decrease in AT levels during cardiac surgery. Positive outcomes associated with maintaining adequate AT levels could be related to its potential beneficial effect on endothelial function (migration and angiogenesis).

A History of the Pharmacological Treatment of Bipolar Disorder.

In this paper, the authors review the history of the pharmacological treatment of bipolar disorder, from the first nonspecific sedative agents introduced in the 19th and early 20th century, such as solanaceae alkaloids, bromides and barbiturates, to John Cade's experiments with lithium and the beginning of the so-called "Psychopharmacological Revolution" in the 1950s. We also describe the clinical studies and development processes, enabling the therapeutic introduction of pharmacological agents currently available for the treatment of bipolar disorder in its different phases and manifestations. Those drugs include lithium salts, valproic acid, carbamazepine, new antiepileptic drugs, basically lamotrigine and atypical antipsychotic agents (olanzapine, risperidone, quetiapine, ziprasidone, aripiprazole, asenapine, cariprazine and lurasidone). Finally, the socio-sanitary implications derived from the clinical introduction of these drugs are also discussed.

RyR2R420Q catecholaminergic polymorphic ventricular tachycardia mutation induces bradycardia by disturbing the coupled clock pacemaker mechanism.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a lethal genetic arrhythmia that manifests syncope or sudden death in children and young adults under stress conditions. CPVT patients often present bradycardia and sino-atrial node (SAN) dysfunction. However, the mechanism remains unclear. We analyzed SAN function in two CPVT families and in a novel knock-in (KI) mouse model carrying the RyR2R420Q mutation. Humans and KI mice presented slower resting heart rate. Accordingly, the rate of spontaneous intracellular Ca2+ ([Ca2+]i) transients was slower in KI mouse SAN preparations than in WT, without any significant alteration in the "funny" current (If ). The L-type Ca2+ current was reduced in KI SAN cells in a [Ca2+]i-dependent way, suggesting that bradycardia was due to disrupted crosstalk between the "voltage" and "Ca2+" clock, and the mechanisms of pacemaking was induced by aberrant spontaneous RyR2- dependent Ca2+ release. This finding was consistent with a higher Ca2+ leak during diastolic periods produced by long-lasting Ca2+ sparks in KI SAN cells. Our results uncover a mechanism for the CPVT-causing RyR2 N-terminal mutation R420Q, and they highlight the fact that enhancing the Ca2+ clock may slow the heart rhythm by disturbing the coupling between Ca2+ and voltage clocks.

Activation of α1A -adrenoceptors desensitizes the rat aorta response to phenylephrine through a neuronal NOS pathway, a mechanism lost with ageing.

BACKGROUND AND PURPOSE: A NO-mediated desensitization of vasoconstrictor responses evoked by stimulation of α1 -adrenoceptors has been reported in different vessels. We investigated the involvement of each α1 -adrenoceptor subtype and constitutive NOS isoforms and the influence of ageing and hypertension on this process. EXPERIMENTAL APPROACH: Wistar and spontaneously hypertensive rats (SHR), 16, 32, 52 and 72 weeks-old, were used to evaluate the desensitization process. Expression of α1 -adrenoceptor subtypes, endothelial NOS (eNOS) and neuronal NOS (nNOS) were determined in rat aorta and left ventricle (LV). Expression levels were also evaluated in LV of a group of heart failure patients with a wide age range. KEY RESULTS: Repeated application of phenylephrine decreased subsequent α1 -adrenoceptor-mediated vasoconstriction by increasing nNOS protein expression in aorta, but not in tail or mesenteric resistance arteries, where mRNA levels of nNOS were undetectable. This desensitization process disappeared in the absence of endothelium or in the presence of L-NAME (100 µM), nNOS inhibitors, SMTC (1 µM) and TRIM (100 µM), and 5-methylurapidil (100 nM, α1A -antagonist), but not BMY7378 (10 nM, α1D -antagonist). The α1A /nNOS-mediated desensitization was absent in aged SHR and Wistar animals, where the expression of α1A -adrenoceptors was reduced in aorta and LV. In human LV, a negative correlation was found between age and α1A -adrenoceptor expression. CONCLUSIONS AND IMPLICATIONS: The α1A -adrenoceptor subtype, through endothelial nNOS-derived NO, may act as a physiological 'brake' against the detrimental effects of excessive α1 -adrenoceptor-mediated vasoconstriction. Reduced α1A -adrenoceptor- and nNOS-mediated desensitization in aged patients could be involved in the age-dependent elevation of adrenergic activity.

Changes in the expression of α1B-adrenoceptor in peripheral mononuclear cells correlates with blood pressure and plasmatic homocysteine.

Human peripheral mononuclear cells (HPMC) have been suggested as a practical surrogate for myocardial or vascular cells. Present work analyses if changes in the expression of α1-adrenoceptors (ARs) in HPMC are related to the hypertensive state and its clinical consequences. Quantitative RT-PCR was employed to evaluate the mRNA levels of the three α1-ARs (α1A, α1B, α1D) in HPMC isolated from normotensive and hypertensive patients, and also in tissues from two animal models of hypertension: spontaneously hypertensive rats (SHR) and hypertension induced by chronic treatment with L-NAME. In patients, 24-h ambulatory blood pressure and serum biochemical profile were also recorded. We found that α1B-AR expression was higher in HPMC from hypertensive patients and correlated with blood pressure and plasmatic homocysteine. A rise in the α1B-AR expression in kidneys, but not in heart from hypertensive animal models was also found. α1D-AR did not change in HPMC, not in rat heart or kidney, but a significant correlation with plasmatic aldosterone was found. In conclusion, we have proved that α1-ARs mRNA expression in HPMC correlates with clinical variables and could be used as a potential biomarker in hypertensive patients.

Investigation of new 2-aryl substituted Benzothiopyrano[4,3-d]pyrimidines as kinase inhibitors targeting vascular endothelial growth factor receptor 2.

Vascular Endothelial Growth Factor (VEGF) pathway has emerged as one of the most important positive modulators of Angiogenesis, a central process implicated in tumour growth and metastatic dissemination. This led to the design and development of anti-VEGF monoclonal antibodies and small-molecule ATP-competitive VEGFR-inhibitors. In this study, we describe the synthesis and the biological evaluation of novel 2-aryl substituted benzothiopyrano-fused pyrimidines 1a-i, 2a-i and 3a-i. The ability of the compounds to target the VEGF pathway was determined in vitro exploiting the compounds' antiproliferative efficacy against HUVEC cells. The VEGFR-2 inhibition was confirmed by enzymatic assays on recombinant human kinase insert domain receptor (KDR), by cell-based phospho-VEGFR-2 inhibition assays, and by ex vivo rat aortic ring tests. The selectivity profile of the best performing derivatives belonging to series 2 was further explored combining modeling studies and additional assays in a panel of human cell lines and other kinases.

Glucocorticoids as modulators of expression and activity of Antithrombin (At): potential clinical relevance.

INTRODUCTION: An inverse relationship has been reported between decreased postoperative Antithrombin (AT) plasmatic levels and the incidence of complications. We hypothesized that Nuclear Hormone Receptors could modulate the expression of SERPINC1, encoding AT, through a Hormone Regulatory Element present in its promoter, and thus hormone analogs could be a pharmacological complement in surgical procedures to activate endogenous AT synthesis. MATERIALS AND METHODS: The expression of SERPINC1 was analyzed in HepG2 cells by quantitative RT-PCR and Western Blot. Two studies were conducted with (a) patients submitted to cardiac surgery with cardiopulmonary bypass receiving (n =17) or not (n=321) glucocorticoids (GCs) as part of their pharmacological treatment, and (b) patients who received (n =20) or not (n=16) GCs as part of their surgery (exodontia or knee arthroscopic, respectively). AT activity in plasma was determined by Innovance Antithrombin Test on a BCS XP System hemostasis analyzer. RESULTS: 13 nuclear hormone receptor ligands were assayed, being GW4064 (FXR ligand) the most potent activator. Retinoids, activating RXR, and GCs (Dexamethasone, cortisone and methylprednisolone) also resulted in increased AT expression. Chronic GC treatment mitigates the decreased AT activity observed after cardiac surgery. In patients who received two acute GC doses, pre-operative and post-operative AT activity was similar, whereas a significant decrease was observed after surgery in untreated patients. CONCLUSIONS: Whereas retinoids and FXR ligands are investigational compounds, regulation of AT by GCS could have a higher potential for translation to clinical practice, pre-conditioning the patient against complications related to reduced AT levels. Larger prospective studies are needed to define the exact role of GCs and their potential clinical utility in cardiac surgery.

Endothelial NT-3 delivered by vasculature and CSF promotes quiescence of subependymal neural stem cells through nitric oxide induction.

Interactions of adult neural stem cells (NSCs) with supportive vasculature appear critical for their maintenance and function, although the molecular details are still under investigation. Neurotrophin (NT)-3 belongs to the NT family of trophic factors, best known for their effects in promoting neuronal survival. Here we show that NT-3 produced and secreted by endothelial cells of brain and choroid plexus capillaries is required for the quiescence and long-term maintenance of NSCs in the mouse subependymal niche. Uptake of NT-3 from irrigating vasculature and cerebrospinal fluid (CSF) induces the rapid phosphorylation of endothelial nitric oxide (NO) synthase present in the NSCs, leading to the production of NO, which subsequently acts as a cytostatic factor. Our results identify a novel interaction between stem cells and vasculature/CSF compartments that is mediated by an unprecedented role of a neurotrophin and indicate that stem cells can regulate their own quiescence in response to endothelium-secreted molecules.

ß-Adrenoceptors differentially regulate vascular tone and angiogenesis of rat aorta via ERK1/2 and p38.

ß-Adrenoceptors (ß-ARs) modulate ERK1/2 and p38 in different cells, but little is known about the contribution of these signaling pathways to the function of ß-ARs in vascular tissue. Immunoblotting analysis of rat aortic rings, primary endothelial (ECs) and smooth muscle cells (SMCs) isolated from aorta showed that ß-AR stimulation with isoprenaline activated p38 in aortic rings and in both cultured cell types, whereas it had a dual effect on ERK1/2 phosphorylation, decreasing it in ECs while increasing it in SMCs. These effects were reversed by propranolol, which by itself increased p-ERK1/2 in ECs. Isoprenaline ß-AR mediated vasodilation of aortic rings was potentiated by the ERK1/2 inhibitor, U0126, in the presence or absence of endothelium or L-NAME, whereas inhibition of p38 had no impact. Isoprenaline moderately decreased sprouting from aorta rings in the Matrigel angiogenesis assay; conversely propranolol not only prevented isoprenaline inhibition, but stimulated angiogenesis. ERK1/2 inhibition decreased angiogenesis, while a dramatic stimulation was observed by p38 blockade. Our results suggest that ERK1/2 activation after ß-ARs stimulation in the smooth muscle hinders the vasodilator effect of isoprenaline, but in the endothelium ß-ARs decreases ERK1/2 and increases p38 activity reducing therefore angiogenesis.

Action of an extract from the seeds of Fraxinus excelsior L. on metabolic disorders in hypertensive and obese animal models.

Nuzhenide and GI3, the principal secoiridoids of an extract obtained from the seeds of Fraxinus excelsior L. (FXE), are believed to be the active compounds responsible for the previously reported hypoglycemic effects of this extract. In this study, the effects of FXE were studied in two animal models which are representative of metabolic disorders: spontaneously hypertensive rats (SHR) and obese Zucker rats. SHR were acutely treated (oral gavage) with different doses of FXE. In addition, SHR and Zucker rats were chronically fed (20 or 5 weeks, respectively) with standard chow supplemented with FXE. Acute treatment with FXE (200 mg per kg body weight) decreased systolic blood pressure as in the case with captopril (50 mg per kg body weight). Chronic treatment with FXE at 100 mg per kg body weight per day, a dose equivalent to that showing hypoglycemic activity in humans, resulted in a significant decrease in glycemia (-16.3%), triglyceridemia (-33.4%) and body weight (-8.1%) in Zucker rats as well as a significant decrease in SBP in SHR (-6.7%), with a concomitant improvement in endothelial function in both strains. The broad-ranging effects of FXE may be due to a unique compositional profile that could be useful to prevent the metabolic syndrome, characterized by obesity, insulin resistance, glucose intolerance, hypertriglyceridemia and elevated blood pressure.