Andrea Emilse Errasti

Bradykinin B1 receptors in human umbilical vein: pharmacological evidence of up-regulation, and induction by interleukin-1β

Bradykinin B1 receptor-mediated responses increase as a function of in vitro incubation in the human umbilical vein. When tissues were continuously treated with the protein synthesis inhibitor, cycloheximide, or with the protein trafficking inhibitor, brefeldin A, pEC50 and maximal response to the selective bradykinin B1 receptor agonist, des-Arg9-bradykinin, were significantly diminished. The anti-inflammatory steroid, dexamethasone, produced a rightward shift of the concentration-response curve to des-Arg9-bradykinin, without affecting the maximal response. Furthermore, lipopolysaccharide or recombinant human interleukin-1 beta potentiate the bradykinin B1-sensitized responses, showing a leftward shift of the concentration-response curve to des-Arg9-bradykinin, without modifying the maximal response. On the other hand, bradykinin B2 receptor-mediated responses were unaffected by continuous exposure to cycloheximide, dexamethasone or lipopolysaccharide. These results provide pharmacological evidence to support the view that the de novo synthesis of bradykinin B1 receptors is involved in the induction of vascular responses in the human umbilical vein. This up-regulation process seems to be selective for bradykinin B1 receptors. The inhibitory effect of dexamethasone and the potentiating actions of lipopolysaccharide and exogenous human recombinant interleukin-1 beta on des-Arg9-bradykinin-mediated responses, suggest the possible role of interleukin-1 beta in the bradykinin B1 receptor up-regulation phenomenon in human umbilical vein.

Targeting aPKC disables oncogenic signaling by both the EGFR and the proinflammatory cytokine TNFα in glioblastoma

Targeting a kinase common to both the EGFR and TNFα signaling pathways may prevent drug resistance in glioblastoma. A New Target in a Deadly Brain Cancer The activity of a growth factor receptor called EGFR is frequently increased in glioblastoma, a common and frequently lethal form of brain cancer. Glioblastoma patients often fail to respond to EGFR kinase inhibitors. Kusne et al. found that the abundance of the kinase aPKC predicted poor prognosis in human glioblastomas. In glioblastoma cells, aPKC was stimulated not only by abnormally active EGFR but also by the proinflammatory cytokine TNFα, which is released by immune cells infiltrating the tumors. EGFR inhibitors were not as effective in suppressing glioblastoma cell growth when administered with TNFα, suggesting that activation of aPKC in response to TNFα could produce resistance to EGFR inhibitors. An inhibitor of aPKC reduced tumor growth in mice with glioblastomas, suggesting that aPKC may be an attractive therapeutic target for glioblastoma treatment because it is downstream of two oncogenic signaling pathways. Grade IV glioblastoma is characterized by increased kinase activity of epidermal growth factor receptor (EGFR); however, EGFR kinase inhibitors have failed to improve survival in individuals with this cancer because resistance to these drugs often develops. We showed that tumor necrosis factor–α (TNFα) produced in the glioblastoma microenvironment activated atypical protein kinase C (aPKC), thereby producing resistance to EGFR kinase inhibitors. Additionally, we identified that aPKC was required both for paracrine TNFα-dependent activation of the transcription factor nuclear factor κB (NF-κB) and for tumor cell–intrinsic receptor tyrosine kinase signaling. Targeting aPKC decreased tumor growth in mouse models of glioblastoma, including models of EGFR kinase inhibitor–resistant glioblastoma. Furthermore, aPKC abundance and activity were increased in human glioblastoma tumor cells, and high aPKC abundance correlated with poor prognosis. Thus, targeting aPKC might provide an improved molecular approach for glioblastoma therapy.

Characterization of α1‐adrenoceptor subtypes mediating vasoconstriction in human umbilical vein

The present study attempted to characterize pharmacologically the subtypes of α‐adrenoceptors mediating contractions in human umbilical vein (HUV). HUV rings were mounted in isolated organ baths and cumulative concentration‐response curves were constructed for the α‐adrenoceptor agonists phenylephrine and adrenaline. Adrenaline was more potent than phenylephrine (pD2=7.29 and 6.04 respectively). Isoproterenol exhibited no agonism on KCl pre‐contracted HUV rings. Propranolol (1 μM) and rauwolscine (0.1 μM) did not affect the concentration‐response curves to adrenaline. These results demonstrate the lack of involvement of functional β‐ or α2‐adrenoceptors in adrenaline‐induced vasoconstriction. The non subtype selective α1‐adrenoceptor antagonist prazosin was evaluated on phenylephrine and adrenaline concentration‐response curves. The effects of the competitive α1A and α1D‐adrenoceptor antagonists, 5‐methyl urapidil and BMY 7378 and the irreversible α1B selective compound chloroethylclonidine (CEC) were also evaluated on adrenaline concentration‐response curves. The potencies of prazosin against responses mediated by adrenaline (pA2=10.87) and phenylephrine (pA2=10.70) indicate the involvement of prazosin‐sensitive functional α1‐adrenoceptor subtype in vasoconstriction of the HUV. The potencies of 5‐methyl urapidil (pA2=6.70) and BMY 7378 (pA2=7.34) were not consistent with the activation of an α1A‐ or α1D‐adrenoceptor population. Exposure to a relatively low CEC concentration (3 μM) abolished the maximum response to adrenaline suggesting that this response was mediated by an α1B‐adrenoceptor subtype. We conclude that HUV express a prazosin‐sensitive functional α1‐adrenoceptor resembling the α1B‐subtype according with the low pA2 values for both 5‐methyl urapidil and BMY 7378 and the high sensitivity to CEC.

MERTK as negative regulator of human T cell activation

The aim of this study was to test the hypothesis whether MERTK, which is up‐regulated in human DCs treated with immunosuppressive agents, is directly involved in modulating T cell activation. MERTK is a member of the TAM family and contributes to regulating innate immune response to ACs by inhibiting DC activation in animal models. However, whether MERTK interacts directly with T cells has not been addressed. Here, we show that MERTK is highly expressed on dex‐induced human tol‐DCs and participates in their tolerogenic effect. Neutralization of MERTK in allogenic MLR, as well as autologous DC–T cell cultures, leads to increased T cell proliferation and IFN‐γ production. Additionally, we identify a previously unrecognized noncell‐autonomous regulatory function of MERTK expressed on DCs. Mer‐Fc protein, used to mimic MERTK on DCs, suppresses nai¨ve and antigen‐specific memory T cell activation. This mechanism is mediated by the neutralization of the MERTK ligand PROS1. We find that MERTK and PROS1 are expressed in human T cells upon TCR activation and drive an autocrine proproliferative mechanism. Collectively, these results suggest that MERTK on DCs controls T cell activation and expansion through the competition for PROS1 interaction with MERTK in the T cells. In conclusion, this report identified MERTK as a potent suppressor of T cell response.

EV-077 in vitro inhibits platelet aggregation in type-2 diabetics on aspirin

INTRODUCTION This study aimed to characterize the in vitro effect of EV-077, a compound that antagonises the binding of prostanoids and isoprostanes to the thromboxane receptor (TP) and inhibits the thromboxane synthase (TS), on platelet aggregation of patients with type-2 diabetes and coronary artery disease (CAD) on chronic aspirin treatment. The effect of EV-077 on 8-iso-PGE(2)-mediated TP receptor contraction of human arteries was also investigated. MATERIALS AND METHODS Fifty-two type-2 diabetics with CAD on chronic aspirin (100 mg) treatment were studied. Arachidonic acid-induced platelet aggregation was measured by impedance aggregometry in platelet-rich plasma (PRP) and whole blood anticoagulated with hirudin, and by light transmission aggregometry in citrate-anticoagulated PRP following 10-min in vitro exposure to EV-077 (100 nmol/l) or control. The effect of EV-077 was measured on isometric contraction of 24 human umbilical arteries induced by isoprostane 8-iso-PGE(2). RESULTS Arachidonic acid (1 mmol/l) induced substantial aggregation in hirudin-anticoagulated whole blood (63 ± 4 AU), which was significantly reduced by in vitro exposure to EV-077 (38 ± 3 AU, P<0.001). Virtually no arachidonic acid-induced aggregation in citrate-anticoagulated or hirudin-anticoagulated PRP was observed. EV-077 potently, competitively and reversibly inhibited TP mediated contraction of umbilical arteries by 8-iso-PGE(2) (P<0.01). CONCLUSIONS Aspirin did not completely inhibit arachidonic acid-induced platelet aggregation in whole blood from type-2 diabetics with CAD. This aggregation is likely induced by prostanoids and/or isoprostanes produced by leukocytes, because it was significantly reduced by EV-077. The TP receptor-mediated contraction of human arteries induced by isoprostane 8-iso-PGE(2) was effectively inhibited by EV-077.

Expression and functional evidence of the prostaglandin F2α receptor mediating contraction in human umbilical vein

Our purposes were to perform the pharmacological characterization of PGF(2alpha) receptor (prostanoid FP-receptor) involved in human umbilical vein contraction and confirm its expression in this tissue. Umbilical cords from healthy patients after full-term deliveries were employed. The vein was dissected out of cords and used for either isolated organ bath or reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assays. The natural prostanoid FP-receptor agonist, PGF(2alpha), and its selective analogues, latanoprost and bimatoprost free acids are full agonists (produce more than 80% of the maximal contractile response to 5-HT) in human umbilical vein. The agonist potency (pEC(50)) order was PGF(2alpha) (6.01+/-0.05)>latanoprost free acid (5.65+/-0.07)=bimatoprost free acid (5.59+/-0.08). The contractile effects of PGF(2alpha) and latanoprost free acid were blocked competitively by the prostanoid FP-receptor antagonist, AL-8810. The antagonist potencies (pK(B)) of AL-8810 vs. PGF(2alpha) (5.93+/-0.05) and vs. latanoprost free acid (6.40+/-0.08) in human umbilical vein are in good agreement with its ability to antagonize prostanoid FP receptors of rat, mouse and human cells. In all samples, clear signal was detected for cDNA amplification of prostanoid FP receptor and the specific prostanoid FP-receptor antibody recognized a protein of approximately 64 kDa. In conclusion, taking into account the obtained functional and biochemical data, we propose for the first time that human umbilical vein express prostanoid FP-receptors and these receptors could be involved in the vasoconstriction action of PGF(2alpha) in this tissue.

Endothelial angiotensin-converting enzyme and neutral endopeptidase in isolated human umbilical vein: an effective bradykinin inactivation pathway.

Kinins are metabolized by metallopeptidases present in different tissues. The aim of this study was to evaluate, employing functional studies in isolated human umbilical vein, the possible participation of angiotensin-converting enzyme, neutral endopeptidase and aminopeptidase P as an inactivation pathway of bradykinin, as well as assess if the endothelial layer is involved in this process. Concentration-response curves to bradykinin were constructed after 120 min incubation period on human umbilical vein rings with and without endothelium and enzymatic inhibitors were applied 30 min before construction of concentration-response curves. The presence of endothelium was confirmed by histological studies. Bradykinin-induced contractile responses were potentiated in human umbilical vein without endothelium when compared to intact tissues. Application of captopril 1 μM (angiotensin-converting enzyme inhibitor) or phosphoramidon 10 μM (neutral endopeptidase inhibitor) induced a leftward shift of bradykinin-elicited responses in human umbilical vein with endothelium while no effect was observed in tissues denuded of endothelium under the same treatment. Exposure to apstatin 10 μM (aminopeptidase P inhibitor) did not potentiate bradykinin-induced effects in intact human umbilical vein. When angiotensin-converting enzyme and neutral endopeptidase were concomitantly inhibited, there was a higher potentiation of bradykinin-elicited responses compared to the effects observed under individual inhibition of either enzyme. Moreover, concentration-response curves to FR190997, a non-peptidic bradykinin B(2) receptor agonist, were not modified under dual enzymatic inhibition. In conclusion, our results demonstrate for the first time the functional relevance of angiotensin-converting enzyme and neutral endopeptidase, localized on the endothelial layer, acting concurrently as a bradykinin inactivating pathway in isolated human umbilical vein.

Neutral Endopeptidase Up-Regulation in Isolated Human Umbilical Artery: Involvement in Desensitization of Bradykinin-Induced Vasoconstrictor Effects

Previous reports show that bradykinin B2 receptors mediate contractile responses induced by bradykinin (BK) in human umbilical artery (HUA). However, although it has been reported that BK-induced responses can desensitize in several inflammatory models, the effects of prolonged in vitro incubation on BK-induced vasoconstriction in HUA have not been studied. In isolated HUA rings, BK-induced responses after a 5-h in vitro incubation showed a marked desensitization compared with responses at 2 h. Inhibition of either angiotensin-converting enzyme (ACE) or neutral endopeptidase (NEP), both BK-inactivating enzymes, failed to modify responses to BK at 2 h. After 5 h, ACE inhibition produced only a slight potentiation of BK-induced responses. In contrast, BK-induced vasoconstriction at 5 h was markedly potentiated by NEP inhibition. Moreover, NEP activity, measured by hydrolysis of its synthetic substrate (Z-Ala-Ala-Leu-p-nitroanilide), showed a 2.4-fold increase in 5-h incubated versus 2-h incubated tissues, which was completely reversed by cycloheximide (CHX) treatment. Furthermore, CHX significantly potentiated BK-induced responses, suggesting that NEP-mediated kininase activity increase at 5 h depends on de novo protein synthesis. In addition, under NEP inhibition, CHX treatment failed to produce an additional potentiation of BK-induced vasoconstriction. Still, NEP up-regulation was confirmed by Western blot, showing a 2.1-fold increase in immunoreactive NEP in 5-h incubated versus 2-h incubated HUA. In summary, the present study provides strong pharmacological evidence that NEP is up-regulated and plays a key role in desensitization of BK-induced vasoconstriction after prolonged in vitro incubation in HUA. Our results provide new insights into the possible mechanisms involved in BK-induced response desensitization during sustained inflammatory conditions.

The endocannabinoid anandamide inhibits kinin B1 receptor sensitization through cannabinoid CB1 receptor stimulation in human umbilical vein

The possible inhibition of kinin B(1) receptor up-regulation by arachidonoylethanolamide (anandamide) was evaluated in isolated human umbilical vein. Anandamide and its metabolically stable analogue, R-N-(2-Hydroxy-1-methylethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (R-(+)-methanandamide), produced a selective and dose-dependent inhibition of kinin B(1) receptor-sensitized contractile responses. The inhibitory effect of anandamide on B(1) receptor-sensitized responses failed to be modified either by 5-biphenyl-4-ylmethyl-tetrazole-1-carboxylic acid dimethylamide (LY2183240), a selective anandamide uptake inhibitor, or 6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-y l](4-methoxyphenyl) methanone (AM630), selective cannabinoid CB(2) receptor antagonist. However, the cannabinoid CB(1) receptor antagonist, N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophen yl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), abolished anandamide effects on kinin B(1) receptor sensitization. The present results provide strong pharmacological evidence indicating that endocannabinoid anandamide inhibits kinin B(1) receptor up-regulation through cannabinoid CB(1) receptor stimulation in human umbilical vein.

CD207+CD1a+ cells circulate in pediatric patients with active Langerhans cell histiocytosis

Langerhans cell histiocytosis (LCH) is a rare disease with an unknown etiology characterized by heterogeneous lesions containing CD207+CD1a+ cells that can arise in almost any tissue and cause significant morbidity and mortality. Precursors of pathological Langerhans cells have yet to be defined. Our aim was to identify circulating CD207+CD1a+ cells and their inducers in LCH. Expression of CD207 and CD1a in the blood myeloid compartment as well as thymic stromal lymphopoietin (TSLP) and transforming growth factor β (TGF-β) plasma levels were measured in 22 pediatric patients with active disease (AD) or nonactive disease (NAD). In patients with AD vs those with NAD, the myeloid compartment showed an increased CD11b (CD11bhigh plus CD11b+) fraction (39.7 ± 3.6 vs 18.6 ± 1.9), a higher percentage of circulating CD11bhighCD11c+CD207+ cells (44.5 ± 11.3 vs 3.2 ± 0.5), and the presence of CD11chighCD207+CD1a+ cells (25.0 ± 9.1 vs 2.3 ± 0.5). Blood CD207+CD1a+ cells were not observed in adult controls or umbilical cord. Increased TSLP and TGF-β levels were detected in patients with AD. Interestingly, plasma from patients with AD induces CD207 expression on CD14+ monocytes. We conclude that CD207+CD1a+ cells are circulating in patients with active LCH, and TSLP and TGF-β are potential drivers of Langerhans-like cells in vivo.