Estrogen, angiogenesis, immunity and cell metabolism: Solving the puzzle

Estrogen plays an important role in the regulation of cardiovascular physiology and the immune system by inducing direct effects on multiple cell types including immune and vascular cells. Sex steroid hormones are implicated in cardiovascular protection, including endothelial healing in case of arterial injury and collateral vessel formation in ischemic tissue. Estrogen can exert potent modulation effects at all levels of the innate and adaptive immune systems. Their action is mediated by interaction with classical estrogen receptors (ERs), ER\u3b1 and ER\u3b2, as well as the more recently identified G-protein coupled receptor 30/G-protein estrogen receptor 1 (GPER1), via both genomic and non-genomic mechanisms. Emerging data from the literature suggest that estrogen deficiency in menopause is associated with an increased potential for an unresolved inflammatory status. In this review, we provide an overview through the puzzle pieces of how 17\u3b2-estradiol can influence the cardiovascular and immune systems

Effects of ω-3 PUFA-enriched diet on growth parameters in a syngenic murine model of breast adenocarcinoma: a possible role for estrogen receptor alpha

Background: Breast cancer (BC) is the most common tumour among women and 75% of BC are estrogen receptor (ER)dependent. In particular, ERα promotes tumour growth, while ERβ has an anti-proliferative effect [1]. Epidemiological data have linked ω-3 polyunsaturated fatty acid (PUFA) consumption to lower incidence of BC and several experimental studies showed the anti-proliferative effects of ω-3 fish oil in different tumour models [2,3]. Chia seed oil is rich in α-linolenic acid (ALA 18:3 ω-3), while corn oil is rich in linoleic acid (LA 18:2 ω-6), precursors of eicosapentaenoic acid (EPA) and arachidonic acid (AA), respectively. Based on substrate availability, these FAs give rise to different eicosanoid signatures with opposite effects in cancer [4]. ω-3 PUFAs generate both anti-inflammatory prostanoids and reactive oxygen species (ROS), which in turn could affect NF-κB. Indeed, NF-κB belongs to a family of transcription factors with a key role in inflammation and oxidative stress, but its role in tumour development is still controversial [5]. The aim of the study was to determine possible processes that are activated by dietary lipids regulating BC growth and metastasis.Fil: Vara Messler, Marianela. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Instituto de Biología Celular; Argentina.Fil: Vara Messler, Marianela. Universidad de Padova. Departamento de Ciencias Farmacéuticas y Farmacológicas; Italia.Fil: Pasqualini, María Eugenia. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Instituto de Biología Celular; Argentina.Fil: Comba, Andrea. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Instituto de Biología Celular. Cátedra de Biología, Histología y Embriología; Argentina.Fil:Comba, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ciencias de la Salud; Argentina.Fil: Toniolo, Alicia. Universidad de Padova. Departamento de Ciencias Farmacéuticas y Farmacológicas; Italia.Fil: Trenti, Annalisa. Universidad de Padova. Departamento de Ciencias Farmacéuticas y Farmacológicas; Italia.Fil: Quiroga, Patricia. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Instituto de Biología Celular; Argentina.Fil: Valentich, Mirta Ana. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Instituto de Biología Celular; Argentina.Fil: Valentich, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ciencias de la Salud; Argentina.Fil: Bolego, Chiara. Universidad de Padova. Departamento de Ciencias Farmacéuticas y Farmacológicas; Italia.Otras Ciencias de la Salu

Role of cerebrospinal fluid biomarkers to predict conversion to dementia in patients with mild cognitive impairment: a clinical cohort study

Abstract Background: Cerebrospinal fluid (CSF) levels assessment of Aβ1-42 and Tau proteins may be accurate diagnostic biomarkers for the differentiation of preclinical Alzheimer's disease (AD) from age-associated memory impairment, depression and other forms of dementia in patients with mild cognitive impairment (MCI). The aim of our study was to explore the utility of CSF biomarkers in combination with common cognitive markers as predictors for the risk of AD development, and other forms of dementia, and the time to conversion in community patients with MCI. Methods: A group of 71 MCI patients underwent neurological assessment, extended neuropsychological evaluation, routine blood tests, ApoE determination, and lumbar puncture to dose t-tau, p-tau181, Aβ1-42. We investigated baseline CSF and neuropsychological biomarker patterns according to groups stratified with later diagnoses of AD conversion (MCI-AD), other dementia (MCI-NAD) conversion, or clinical stability (sMCI). Results: Baseline Aβ1-42 CSF levels were significantly lower in MCI-AD patients compared to both sMCI and MCI-NAD. Additionally, p-tau181 was higher in the MCI-AD group compared to sMCI. The MCI-AD subgroup analysis confirmed the role of Aβ1-42 in its predictive role of time to conversion: rapid converters had lower Aβ1-42 levels compared to slow converters. Logistic regression and survival analysis further supported the key predictive role of baseline Aβ1-42 for incipient AD and dementia-free survival. Conclusions: Our results confirm the key role of CSF biomarkers in predicting patient conversion from MCI to dementia. The study suggests that CSF biomarkers may also be reliable in a real world clinical setting

The Glycolytic Enzyme PFKFB3 Is Involved in Estrogen-Mediated Angiogenesis via GPER1 s

ABSTRACT The endogenous estrogen 17b-estradiol (E2) is a key factor in promoting endothelial healing and angiogenesis. Recently, proangiogenic signals including vascular endothelial growth factor and others have been shown to converge in endothelial cell metabolism. Because inhibition of the glycolytic enzyme activator phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3) reduces pathologic angiogenesis and estrogen receptor (ER) signaling stimulates glucose uptake and glycolysis by inducing PFKFB3 in breast cancer, we hypothesized that E2 triggers angiogenesis in endothelial cells via rapid ER signaling that requires PFKFB3 as a downstream effector. We report that treatment with the selective G protein-coupled estrogen receptor (GPER1) agonist G-1 (10 210 to 10 27 M) mimicked the chemotactic and proangiogenic effect of E2 as measured in a number of short-term angiogenesis assays in human umbilical vein endothelial cells (HUVECs); in addition, E2 treatment upregulated PFKFB3 expression in a time-and concentrationdependent manner. Such an effect peaked at 3 hours and was also induced by G-1 and abolished by pretreatment with the GPER1 antagonist G-15 or GPER1 siRNA, consistent with engagement of membrane ER. Experiments with the PFKFB3 inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one showed that PFKFB3 activity was required for estrogen-mediated HUVEC migration via GPER1. In conclusion, E2-induced angiogenesis was mediated at least in part by the membrane GPER1 and required upregulation of the glycolytic activator PFKFB3 in HUVECs. These findings unravel a previously unrecognized mechanism of estrogen-dependent endocrine-metabolic crosstalk in HUVECs and may have implications in angiogenesis occurring in ischemic or hypoxic tissues

Optimization of Laboratory Diagnostics of Primary Biliary Cholangitis: When Solid-Phase Assays and Immunofluorescence Combine

The laboratory diagnostics of primary biliary cholangitis (PBC) have substantially improved, thanks to innovative analytical opportunities, such as enzyme-linked immunosorbent assays (ELISA) and multiple immunodot liver profile tests, based on recombinant or purified antigens. This study aimed to identify the best diagnostic test combination to optimize PBC diagnosis. Between January 2014 and March 2017, 164 PBC patients were recruited at the hospitals of Parma, Modena, Reggio-Emilia, and Piacenza. Antinuclear antibodies (ANA) and anti-mitochondrial antibodies (AMA) were assayed by indirect immunofluorescence (IIF), ELISA, and immunodot assays (PBC Screen, MIT3, M2, gp210, and sp100). AMA-IIF resulted in 89.6% positive cases. Using multiple immunodot liver profiles, AMA-M2 sensitivity was 94.5%, while anti-gp210 and anti-sp100 antibodies were positive in 16.5% and 17.7% of patients, respectively. PBC screening yielded positive results in 94.5% of cases; MIT3, sp100, and gp210 were detected by individual ELISA test in 89.0%, 17.1%, and 18.9% of patients, respectively. The association of PBC screening with IIF-AMA improved the diagnostic sensitivity from 89.6% to 98.2% (p < 0.01). When multiple immunodot liver profile testing was integrated with AMA-IIF, the diagnostic sensitivity increased from 89.1% to 98.8% (p < 0.01). The combination of IIF with solid-phase methods significantly improved diagnostic efficacy in PBC patients

Bisdemethoxycurcumin and Its Cyclized Pyrazole Analogue Differentially Disrupt Lipopolysaccharide Signalling in Human Monocyte-Derived Macrophages

Several studies suggest that curcumin and related compounds possess antioxidant and anti-inflammatory properties including modulation of lipopolysaccharide- (LPS-) mediated signalling in macrophage cell models. We here investigated the effects of curcumin and the two structurally unrelated analogues GG6 and GG9 in primary human blood-derived macrophages as well as the signalling pathways involved. Macrophages differentiated from peripheral blood monocytes for 7 days were activated with LPS or selective Toll-like receptor agonists for 24 h. The effects of test compounds on cytokine production and immunophenotypes evaluated as CD80+/CCR2+ and CD206+/CD163+ subsets were examined by ELISA and flow cytometry. Signalling pathways were probed by Western blot. Curcumin (2.5–10 μM) failed to suppress LPS-induced inflammatory responses. While GG6 reduced LPS-induced IκB-α degradation and showed a trend towards reduced interleukin-1β release, GG9 prevented the increase in proinflammatory CD80+ macrophage subset, downregulation of the anti-inflammatory CD206+/CD163+ subset, increase in p38 phosphorylation, and increase in cell-bound and secreted interleukin-1β stimulated by LPS, at least in part through signalling pathways not involving Toll-like receptor 4 and nuclear factor-κB. Thus, the curcumin analogue GG9 attenuated the LPS-induced inflammatory response in human blood-derived macrophages and may therefore represent an attractive chemical template for macrophage pharmacological targeting

Analysis of the molecular mechanisms of the antineoplastic effect of ouabain

Ouabain is a cardiac glycoside whose primary action is inhibition of Na/K ATPase activity, a ubiquitous enzyme responsible for translocating Na and K ions across the cell membrane using ATP as the driving force. It has been demonstrated that the Na/K ATPase also functions as a classical receptor, capable of converting cardiac glycodise binding into activation of various protein kinase cascades (Liu and Xie, 2010). Also, recent studies have shown that cardiac glycosides selectively inhibit cell proliferation and/or induce apoptosis in several cancer cell lines (Schoner and Sheiner-Bobis, 2007). These in vitro studies are supported by epidemiological data reporting a protection from several types of cancer in patients who were on cardiac glycoside treatment (Stenkvist, 1999; Haux 1999; Haux et al, 2001). To elucidate the cellular and molecular mechanisms underlying cardiac glycoside effect against cancer cells, we studied the effect of ouabain (1-100 nM) on two cancer cell lines, Jurkat (human T cell lymphoblast-like cell line) and A549 (human lung adenocarcinoma epithelial cell line). Ouabain (1-20 nM) induced a concentration-dependent decrease in proliferation of both cell lines. At higher concentrations (50-100 nM) ouabain was cytotoxic for the two cancer cell lines, as demonstrated by the increase in Annexin V/propidium iodide binding. At the same concentrations, ouabain did not affect cell viability of peripheral blood mononuclear cells (PBMC) and, as previously shown in our laboratory, protected from apoptosis human umbilical vein endothelial cells (HUVEC) (Trevisi L et al., 2004). Ouabain increased Src and ERK1/2 phosphorylation in A549 and Jurkat thus demonstrating the activation of the known signaling pathways triggered by Na/K ATPase signalosome. However, pharmacological inhibition of Src or MEK did not abolish the cytotoxic activity of ouabain. In both cell lines ouabain caused a decrease of phospho-Akt levels. Decrease of Bcl-2 protein levels and mitochondrial membrane potential were early events of ouabain treatment in both cell lines. However, only in Jurkat cells were observed caspase 3/7 activation, DNA ladder fragmentation and inhibition of cell death by the pan caspase inhibitor Z-VAD-fmk, hallmarks of caspase-dependent apoptosis. On the other hand, a marker of autophagy, i.e. conversion of LC3-I to LC3-II isoform, was observed in ouabain-treated A549. Moreover, ouabain enhanced the autophagic flux in A549 as demonstrated by increase of p62 degradation and increase of punctate pattern of LC3 after co-incubation with chloroquine, a drug that blocks fusion of autophagosomes with lysosomes. Furthermore, cell death induced by ouabain was completely blocked by treatment with an inhibitor of autophagy such as 3-methyladenine. It has been suggested that decrease of Bcl-2 levels could induce autophagy since Bcl-2 interacts with the autophagic protein Beclin 1, inhibiting the formation of the autophagy initiation complex (Pattingre S et al., 2005). This interaction is regulated by JNK because JNK-dependent phosphorylation of Bcl-2 causes Bcl-2 degradation and disruption of Bcl-2/Beclin1 complex. In A549 treated with ouabain the levels of Beclin 1 were manteined costant. However, inhibition of JNK with SP600125 blocked cell death induced by ouabain only in A549. We speculate that JNK activation by ouabain in A549 reduces Bcl-2 levels thus releasing Beclin 1 and inducing autophagy. Further studies will be required to confirm this hypothesis. Finally, to ascertain whether differences in sensitivity to ouabain among normal and cancer cells are related to a specific pattern of Na/K ATPase α subunit isoform expression, western blotting analysis of α isoforms was performed in Jurkat and A549 cells and compared to the expression pattern of two normal cell lines: HUVEC and PBMC. We found that α1 isoform is ubiquitous, α2 isoform is not expressed only in PBMC and that α3 isoform is expressed exclusively in the two cancer cell lines. These data suggest that α3 subunit could be critical for ouabain cytotoxic effect.L’ouabaina é un glicoside cardioattivo la cui azione più nota è l’inibizione della Na/K ATPasi, enzima ubiquitario della membrana plasmatica responsabile del trasporto di ioni Na e K attraveso le membrane utilizzando ATP come forza motrice. È stato dimostrato che la Na/K ATPasi, oltre alla sua funzione di pompa, é in grado di agire come recettore, attivando varie cascate di segnale (Liu and Xie, 2010). Studi recenti hanno dimostrato che i glicosidi cardioattivi inibiscono in maniera selettiva la proliferazione e/o inducono apoptosi in diverse linee di cellule tumorali (Schoner and Sheiner-Bobis, 2007). Questi studi in vitro sono supportati da dati epidemiologici che evidenziano una protezione da vari tipi di cancro in pazienti trattati con glicosidi cardioattivi (Stenkvist, 1999; Haux 1999; Haux et al, 2001). Al fine di chiarire il meccanismo molecolare e cellulare alla base dell’effetto citotossico dei glicosidi cardioattivi sulle cellule tumorali, abbiamo studiato gli effetti dell’ouabaina (1-100 nM) nei confronti di due linee cellulari tumorali, le Jurkat (una linea proveniente da un linfoma a cellule T) e le A549 (linea ottenuta da carcinoma bronchiolo-alveolare). Il trattamento con ouabaina (1-20 nM) provocava una diminuzione della proliferazione cellulare concentrazione dipendente in entrambe le linee cellulari. Alle concentrazioni più elevate (50-100 nM) l’ouabaina era citotossica per le due linee cellulari testate, come dimostrato dall’aumento di cellule Annessina V/propidio positive. Alle stesse concentrazioni l’ouabaina non alterava la vitalità delle cellule mononucleate estratte da sangue periferico umano (PBMC) e, come già dimostrato nel nostro laboratorio, proteggeva dall’apoptosi le cellule endoteliali della vena ombelicale umana (HUVEC) (Trevisi L et al., 2004). Il trattamento con ouabaina induceva l’attivazione delle chinasi Src ed ERK1/2 in entrambe le linee cellulari, confermando così la stimolazione della nota cascata di segnale innescata dala Na/K ATPasi. Tuttavia, l’inibizione farmacologica della Src o della MEK non contrastava l’effetto citotossico dell’ouabaina. In entrambe le linee cellulari il trattamento con ouabaina induceva una diminuzione della fosforilazione di Akt. In entrambe le linee cellulari, la diminuzione della proteina antiapoptotica Bcl-2 e del potenziale di membrana mitocondriale costituivano gli eventi iniziali a seguito del trattamento con ouabaina. Tuttavia, solo nelle Jurkat sono stati riscontrati alcune marcatori dell’apoptosi caspasi-dipendente quali l’attivazione delle caspasi 3/7, la tipica frammentazione del DNA (DNA ladder) e l’inibizione della morte cellulare indotta dall’inibitore pan caspasico Z-VAD-fmk. Al contrario nelle A549 sono stati riscontrati i classici marcatori di autofagia come la conversione di LC3 dalla forma LC3-I alla isoforma LC3-II. Inoltre l’ouabaina aumentava il flusso autofagico nelle A549, come dimostrato dall’aumento della degradazione di p62 e dall’aumento delle forme puntate di LC3 in caso di co-incubazione con clorochina, un inibitore della fusione lisosomi-autofagosomi. Infine la morte cellulare indotta da ouabaina era bloccata dal trattamento con l’inibitore dell’autofagia 3-metiladenina. È stato ipotizzato che la diminuzione della Bcl-2 induca autofagia in quanto la Bcl-2 interagisce con Beclin 1 prevenendo così la formazione del complesso di iniziazione (Pattingre S et al., 2005). Tale interazione è regolata da JNK, la quale fosforilando Bcl-2, causa la sua degradazione e la rottura del complesso Bcl-2/Beclin 1. Nelle A549 trattate con ouabaina i livelli di Beclin 1 erano costanti, ma l’inibizione di JNK con SP600125 bloccava la morte cellulare. Noi ipotizziamo che l’attivazione di JNK da parte dell’ouabaina nelle A549 riduca i livelli di Bcl-2 permettendo il rilascio di Beclin 1 e di conseguenza attivi il processo di autofagia. Ulteriori studi saranno necessari per chiarire questo punto. Infine, attraverso l’analisi western blotting è stata studiata l’espressione delle varie isoforme della subunità α della Na/K ATPase nelle due linee tumorali studiate (A549 e Jurkat) e paragonata a quella di due linee normali (HUVEC e PBMC), allo scopo di verificare se le differenze nella sensibilità all’ouabaina fra cellule normali e tumorali possano essere ascritte ad uno specifico tipo di espressione della varie isoforme della subunità α. I nostri risultati indicano che l’isoforma α1 è ubiquitaria, l’isoforma α2 non è espressa solo nei PBMC mentre l’isoforma α3 è espressa solo nelle due linee cellulari tumorali. Questo dato suggerisce che la subunità α3 possa essere determinante per gli effetti citotossici dei glicosidi cardioattivi nei confronti delle cellule tumorali

Signalling pathways involved in the apoptotic action of ouabain in two different cancer cell lines

Cardiac glycosides, like ouabain, are specific inhibitors of plasma membrane Na+/K+-ATPase, enzyme responsible for translocating Na+and K+ ions across cell membrane using ATP as energy source. Recent findings suggest for Na+/K+-ATPase a role as signal transducer, involved in the control of cell proliferation and growth or in the protection against apoptotic stimuli. In fact, the binding of ouabain to Na+/K+-ATPase (at concentrations that do not inhibit the pump activity) triggers a complex signaling cascade that is initiated by interacting with neighboring membrane proteins and organized cytosolic cascades of signaling molecules. These signaling complexes send messages to intracellular organelles via the activation of the protein tyrosine kinase Src, transactivation of epidermal growth factor receptor (EGFR) by Src, activation of Ras and the extracellular signal-regulated kinase (ERK). Also a ROS dependent c-Jun N-terminal kinase (JNK) activation has been shown to be involved in the pathways activated by ouabain (1). In agreement with these findings, we have previously showed that ouabain has an antiapoptotic effect on HUVEC through the activation of phosphoinositide-3 kinase (PI3K) and ERK (2). On the other hand, several studies have suggested that cardiac glycosides may have an anticancer utilization. Evidences of the antineoplastic potential of cardiac glycosides have been obtained with in vitro studies, but the most relevant evidences of the beneficial effects of cardiac glycosides in cancer treatment were drawn from epidemiological data. The death rate and cancer recurrence turned out to be lower in women with breast cancer treated with digitalis than in non-treated patients. Moreover, it was observed a reduced incidence of leukemia/lymphoma and kidney/urinary tract tumours in subjects with elevated plasmatic concentrations of digitoxin (3). The aim of my doctoral work is to characterize the effect of nM concentrations of ouabain on two cancer cell lines: Jurkat (immortalized cell line of T lymphocytes) and A549 (carcinomic human alveolar basal epithelial cells) with particular attention to the signaling pathway involved. Cell treatment with ouabain (1-100 nM) for 24 h induced a concentration-dependent decrease in cell viability measured by the MTT reduction assay both in Jurkat and A549 cells. The decrease in cell viability at 100 nM was 67.0\ub12.2 e 70.0\ub12.1 in Jurkat and A549, respectively. Incubation of both cell lines with 100 nM ouabain for 24 h induced a significant raise in the number of apoptotic cells, as indicated by flow cytometric analysis of annexin V/propidium iodide binding: ouabain increased the proportion of annexin V positive (apoptotic) cells from 7.0\ub10.7% to 33.0\ub16.0% in Jurkat cells and from 7.0\ub10.6% to 22.0\ub12.0% in A549 cells. However, an increase in caspase-3 activation induced by ouabain treatment was observed only in Jurkat cells. In order to clarify the signaling pathways involved in the mechanism of action of ouabain, the role of ERK, Src kinase and JNK was investigated. A transient increase in ERK1/2 phosphorylation (determined by Western blotting analysis) was observed in Jurkat cells treated with 100 nM ouabain for 30 min. Nevertheless, incubation of the cells with a specific MEK inhibitor, PD98059 (25 \u3bcM) or U0126 (10 \u3bcM) did not abolish the apoptotic effect of ouabain, as shown by MTT test and flow cytometric analysis of annexin V/PI binding. Similar results were obtained with A549 cells. Furthermore, treatment of both tumour cell lines with the inhibitor of JNK, SP600125, did not affect the apoptotic action of ouabain. To ascertain whether the activation of Src kinase is required for the ouabain-effect on cancer cells we used the Src-kinase inhibitor PP2 (50 \u3bcM). This compound did not affect the ouabain-induced apoptosis in both Jurkat and A549 cells. Our results show that nM concentrations of ouabain are pro-apoptotic for Jurkat and A549 cells through a mechanism that does not involve the classical pathways of signal transduction activated by ouabain in non-tumoral cells. References Schoner W et al. (2007) Am J Physiol Cell Physiol 293:509 Trevisi L et al. (2004) Biochem Biophys Res Commun 321:716 Newman RA et al. (2008) Mol Interv 8:3

Differential post-transcriptional regulation of COX-2 expression in human umbilical vein endothelial cells derived from diabetic and healthy women: role of microR-As

COX-2 is one of the vasoprotective genes up-regulated by steady laminar shear stress (Topper et al. 1996; Di Francesco et al. 2009) and produces the vasoprotective prostacyclin (Grosser et al. 2006; Di Francesco et al. 2009). Early investigations have shown that diabetes markedly alters prostanoid synthesis in the vasculature (Bagi et al. 2006) but studies on the potential involvement of COX-2 in diabetic vascular complications have given controversial results. The aim of this study was to investigate whether the exposure to a diabetic environment in vivo could affect the regulation of endothelial COX-2 expression through post-transcriptional mechanisms. Thus, we compared COX-2 expression in human umbilical vein endothelial cells derived from normal (nHUVEC) and type I diabetic mothers (dHUVEC). Confluent monolayers of cells at passage level 3 were treated with or without IL-1\u3b2 5 ng/ml for 6 or 24 h in medium 199/DMEM (50:50) supplemented with ECGF, pen-strep, glutamine, and 5% FCS. The medium was assayed for 6-keto-PGF1\u3b1, PGE2, PGF2\u3b1, PGD2 by radioimmunoassay or ELISA, cell lysates for COX-1, COX-2, microsomal PGES-1 (mPGES-1), prostacyclin synthase (PGIS), and heme oxygenase (HO)-1 by specific Western blot techniques. RNAs were extracted and analysed for COX-2, microRNA(miR)542-3p, and miR16 by real time-PCR. In dHUVEC there was a statistically significant increase in the biosynthesis of 6-keto-PGF1\u3b1, PGE2, and PGD2, but not PGF2\u3b1, in response to IL-1\u3b2 vs nHUVEC. The values of prostanoid generation are reported in the table. Prostanoid levels measured in nHUVEC and dHUVEC after IL-1\u3b2 (5 ng/ml) for 6 and 24 h (values are reported as ng, mean\ub1SEM, n=5-7; *P<0.05 vs nHUVEC) 6-keto-PGF1\u3b1 PGE2 PGF2\u3b1 PGD2 6 h 24 h 6 h 24 h 6 h 24 h 6 h 24 h nHUVEC 2.8 \ub10.5 13.7\ub12.1 3.0\ub10.7 9.2\ub13.0 22.7\ub16.9 122.4\ub129.8 0.6\ub10.1 1.1\ub10.3 dHUVEC 9.9\ub13.6* 37.7\ub110.5* 5.4\ub11.0 23.2\ub15.2* 35.2\ub114.7 132.1\ub154.6 0.8\ub10.3 2.4\ub10.7* In both types of HUVEC, prostanoids were generated by COX-2; in fact, pretreatment with 1 \u3bcM NS-398 (a selective inhibitor of COX-2) caused an almost complete inhibition of prostanoid biosynthesis. After IL-1\u3b2 treatment, Western blot analysis showed that COX-1 and PGIS protein levels were comparable in nHUVEC and dHUVEC while mPGES-1 levels were undetectable both in nHUVEC and dHUVEC. The protein levels of COX-2 were significantly (P<0.05) higher in dHUVEC vs nHUVEC at 24 h (COX-2/\u3b2-actin optical density 2.30\ub10.50 vs 0.88\ub10.12, respectively). mRNA levels of COX-2 in response to IL-1\u3b2 were significantly (P<0.01) higher in dHUVEC than in nHUVEC (17.98\ub11.50 vs 5.91\ub10.60 at 6 h; 9.16\ub10.60 vs 3.46\ub10.70 at 24 h, respectively). Experiments of mRNA stability performed in the presence of actynomycin D (0.65 \u3bcg/ml), to inhibit transcription, demonstrated that COX-2 mRNA was more stable in dHUVEC than in nHUVEC after IL-1\u3b2 stimulation; in fact, at 3h there was 30% vs 13% of COX-2 mRNA remaining in dHUVEC and nHUVEC, respectively (P<0.01). We analysed the levels of two different miRNAs involved in the destabilization of COX-2 mRNA through their binding in the 3\u2019UTR: miR542-3p and miR16. In nHUVEC at 24 h with IL-1\u3b2, miR542-3p and miR16 levels were significantly (P<0.05) increased vs unstimulated cells (relative miRNA levels: miR542-3p, 2.2\ub10.8 vs 1.00\ub10.02; miRNA 16, 1.71\ub10.26 vs 1.04\ub10.02, respectively). On the contrary, in dHUVEC stimulated with IL-1\u3b2 for 24 h, miR542-3p and miR16 levels were lower than in unstimulated cells. Interestingly, miR542-3p and miR16 levels were lower in dHUVEC vs nHUVEC, at 24 h with IL1\u3b2 (0.53\ub10.20 and 0.74\ub10.20, respectively; P<0.05). HO-1 expression was higher (P<0.01) in HUVEC from diabetic than healthy women, in response to IL-1\u3b2 and NS- 398 reduced HO-1 levels both in nHUVEC and dHUVEC. In summary, IL-1\u3b2 induces COX-2-dependent prostanoids in HUVEC. Prostacyclin is a dominant autocrine prostanoid. HUVEC exposed to a diabetic environment express higher levels of COX-2 through post-transcriptional mechanisms. Loss of IL-1\u3b2- dependent inducibility of miRNA-542-3p and miR-16 in dHUVECs is associated with enhanced COX-2 whose expression was associated with higher levels of HO-1. In conclusion, targeting of miRNAs in endothelial cells may represent a new therapeutic strategy to modulate COX-2-dependent prostacyclin. References Topper JN et al. (1996) Proc Natl Acad Sci U.S.A. 93:10417 Di Francesco L et al. (2009) Circ Res 104:506 Grosser T et al. (2006) J Clin Invest 116:4 Bagi Z et al. (2006) Pharmacol Rep 58 Suppl:5