Mouse aortic muscle cells respond to oxygen following cytochrome P450 3A13 gene transfer

We have previously shown that a cytochrome P450 (CYP450) hemoprotein from the 3A subfamily CYP3A13 for the mouse, serves as the sensor in the contraction of the ductus arteriosus in response to increased oxygen tension. In addition, we have identified endothelin-1 (ET-1) as the effector for this response. Here, we examined whether Cyp3a13 gene transfer confers oxygen sensitivity to cultured muscle cells from mouse aorta. Coincidentally, we determined whether the same hemoprotein is normally present in the vessel. Cyp3a13-transfected aortic cells responded to oxygen, whereas no significant response was seen in native cells or in cells transfected with an empty vector. Furthermore, this oxygen effect was curtailed by the ET-1/ETA receptor antagonist BQ-123. We also found that CYP3A13 occurs naturally in aortic tissue and its isolated muscle cells in culture. We conclude that CYP3A13 is involved in oxygen sensing, and its action in the transfected muscle cells of the aorta, as in the native cells of the ductus, takes place through a linkage to ET-1. However, the response of aortic muscle to oxygen, conceivably entailing the presence of CYP3A13 at some special site, is not seen in the native situation, and may instead unfold upon transfection of the parent gene

Evaluation of analytical performance and comparison of clinical results of the new generation method AccuTnI+3 for the measurement of cardiac troponin I using both patients and quality control plasma samples

The study aims are to evaluate the analytical performance and the clinical results of the chemiluminescent Access AccuTnI+3 immunoassay for the determination of cardiac troponin I (cTnI)with DxI 800 and Access2 platforms and to compare the clinical results obtained with this method with those of three cTnI immunoassays, recently introduced in the European market. The limits of blank (LoB), detection (LoD), and quantitation (LoQ) at 20% CV and 10% CV were 4.5 ng/L and 10.9 ng/L, 17.1 and 30.4 ng/L, respectively. The results of STAT Architect high Sensitive TnI (Abbott Diagnostics), ADVIA Centaur Troponin I Ultra (Siemens Healthcare Diagnostics), ST AIA-Pack cTnI third generation (Tosoh Bioscience), and Access AccuTnI + 3 (Beckman Coulter Diagnostics) showed very close correlations (R ranging from 0.901 to 0.994) in 122 samples of patients admitted to the emergency department. However, on average there was a difference up to 2.4-fold between the method measuring the highest (ADVIA method) and lowest cTnI values (AccuTnI + 3 method). The consensus mean values between methods ranged from 6.2% to 29.6% in 18 quality control samples distributed in an external quality control study (cTnI concentrations ranging from 29.3 ng/L to 1557.5 ng/L). In conclusion, the results of our analytical evaluation concerning the AccuTnI + 3 method, using the DxI platform, are well in agreement with those suggested by the manufacturer as well as those reported by some recent studies using the Access2 platform. Our results confirm that the AccuTnI + 3 method for the Access2 and DxI 800 platforms is a clinically usable method for cTnI measurement

Gene silencing of endothelial von Willebrand Factor attenuates angiotensin II-induced endothelin-1 expression in porcine aortic endothelial cells.

Expression of endothelin (ET)-1 is increased in endothelial cells exposed to angiotensin II (Ang II), leading to endothelial dysfunction and cardiovascular disorders. Since von Willebrand Factor (vWF) blockade improves endothelial function in coronary patients, we hypothesized that targeting endothelial vWF with short interference RNA (siRNA) prevents Ang II-induced ET-1 upregulation. Nearly 65 ± 2% silencing of vWF in porcine aortic endothelial cells (PAOECs) was achieved with vWF-specific siRNA without affecting cell viability and growth. While showing ET-1 similar to wild type cells at rest, vWF-silenced cells did not present ET-1 upregulation during exposure to Ang II (100 nM/24 h), preserving levels of endothelial nitric oxide synthase activity similar to wild type. vWF silencing prevented AngII-induced increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity and superoxide anion (O2-) levels, known triggers of ET-1 expression. Moreover, no increase in O2- or ET-1 levels was found in silenced cells treated with AngII or NOX-agonist phorbol ester (PMA 5 nM/48 h). Finally, vWF was required for overexpression of NOX4 and NOX2 in response to AngII and PMA. In conclusion, endothelial vWF knockdown prevented Ang II-induced ET-1 upregulation through attenuation of NOX-mediated O2- production. Our findings reveal a new role of vWF in preventing of Ang II-induced endothelial dysfunction

Valutazione delle prestazioni analitiche di un nuovo metodo per il dosaggio dell'aldosterone

In questi ultimi anni sono stati sviluppati kit commerciali per il dosaggio dell’aldosterone utilizzabili su piattaforme automatizzate. Lo scopo del presente studio è stato valutare le caratteristiche analitiche ed i risultati clinici ottenuti con un nuovo metodo di misura dell’aldosterone che si avvale della piattaforma automatizzata LIAISON (DiaSorin, Saluggia – VC) e confrontare i valori di aldosterone misurati con questo nuovo metodo con quelli ottenuti, sugli stessi campioni, con il metodo RIA ALDO-CTK (DiaSorin, Saluggia - VC), con il metodo automatizzato che utilizza la piattaforma iSYS (IDS, Boldon, UK - distributore italiano: Pantec srl, Torino) e con la spettrometria di massa (metodica Perkin-Elmer). Sono stati anche misurati con questo nuovo metodo 5 campioni di controllo, distribuiti nella VEQ Immunocheck (QualiMedLab srl, Pisa) e i valori trovati sono stati confrontati con quelli ottenuti con la media di consenso dei laboratori partecipanti. A questo scopo sono stati reclutati 147 soggetti apparentemente sani e 199 pazienti con malattie cardiovascolari. Il metodo di dosaggio dell’aldosterone che utilizza la piattaforma automatizzata LIAISON presenta valori di LoB e LoD rispettivamente di 18 pg/mL e 30 pg/mL; mentre i valori di LoQ al 10% CV e al 20% CV corrispondono a concentrazioni di aldosterone pari a 95 pg/mL e 40 pg/mL, rispettivamente. Il metodo automatizzato LIAISON presenta una migliore praticabilità sperimentale rispetto al metodo RIA; in particolare il tempo di analisi è inferiore ad un’ora per il metodo automatizzato, mentre con il metodo RIA occorrono circa 18-24 ore. Infine, il metodo LIAISON presenta risultati clinici che sono confrontabili con quelli ottenuti con il metodo automatizzato che utilizza la piattaforma iSYS (R=0,906, N=294), con quelli della spettrometria di massa (R=0,746, N=30), e con i valori ottenuti in 5 campioni di controllo della VEQ Immunocheck (R=0,936, p= 0,0191). In conclusione, il metodo di dosaggio dell’aldosterone che utilizza la piattaforma automatizzata LIAISON sembra essere preferibile ai metodi manuali RIA ed EIA, soprattutto in quei laboratori, che già utilizzano la piattaforma LIAISON per il dosaggio di altri analiti e che devono eseguire il dosaggio di questo ormone in pochi campioni ogni giorno, ed inoltre fornire al clinico una risposta in tempi brevi

Exosomes released from sulforaphane-treated fibroblasts protect the cardiomyocytes from angiotensin II-induced hypertrophy

Introduction: The communication between fibroblasts and cardiomyocytes underlies the pathological cardiac hypertrophy induced by angiotensin-II (AngII), which contributes to heart failure. Fibroblast-derived exosomes (F-Exo) have been implicated in mediating AngII-induced cardiomyocyte hypertrophy. However, how release of anti-hypertrophic F-Exo is induced, remains an unanswered issue. Sulforaphane (SFN), a naturally occurring isothiocyanate extracted from cruciferous vegetables, attenuates AngII-induced cardiomyocytes hypertrophy. We tested the effects of SFN on the release of anti-hypertrophic F-Exo in vitro. Methods: Murine embryo fibroblasts were treated with non-toxic dose of SFN (3 μM/7 days). Intact F-Exo were isolated from cell culture media by differential centrifugation. F-Exo were quantified by Western blot using CD63. Hypertrophy of HL-1 cardiomyocytes was induced by AngII (100 nM/12 h). Cell viability was assessed by MTT assay. Cell surface area, an indicator of cell hypertrophy, was measured after 3 or 24 h incubation with 30 μg exosomes isolated from SFN-treated (SFN-F-Exo) or untreated (F-Exo, control) fibroblasts. Uptake by HL-1 of DiA-labeled exosomes was measured under rest or AngII. Exosomal content of Maspin, a protease inhibitor with function of inhibitor of histone deacetylase 1, was assessed by Western blot. Results: Treatment with F-Exo significantly increased HL-1 viability by 53% under stress compared to control. Stressed HL-1 treated for 24 h with SFN-F-Exo displayed cell surface area similar to resting cells, but not those treated with F-Exo. Stressed HL-1 exhibited a ~3-fold increase in SFN-F-Exo uptake rather than F-Exo. SFN-F-Exo are enriched in Maspin. Summary/conclusion: SFN increases the uptake of F-Exo which display the ability to prevent AngII-induced cardiomyocytes hypertrophy. Higher content of Maspin in SFN-F-Exo suggests that modulation of exosomal uptake and hypertrophy in stressed cardiomyocytes may be epigenetically driven

Epigenetic regulation of myocardial homeostasis, self-regeneration and senescence

The adult myocardium has limited capacity to preserve, renew or rejuvenate itself. The local microenvironment may induce epigenetic changes affecting the survival, proliferation, function and senescence of cardiac cells at rest and following the exposure to different stressors. The cellular response to microenvironment is characterized by the release of ions, oxygen free radicals, auto/paracrine factors and RNAs that drive the magnitude of gene reprogramming through the interaction with specific promoters. The epigenetic alterations may act at transcriptional and post-transcriptional level and change cardiac physiological traits. The abnormal DNA methylation underlies the progressive decay of contractile function and the angiogenic ability; while, the histone acetylation promotes the survival, function and proliferation of cardiac cells in the presence of ischemic microenvironment. At least, the expression and secretion of microRNAs and long noncoding RNAs may regulate the threshold to stress tolerance of adult cardiac cells and induce the matrix turnover as well. Natural or synthetic active compounds effectively modulate the epigenetic state of cardiac cells. Plant foods contain many active compounds with epigenetic properties and might assume a clinical significance as natural cardiac regenerators or rejuvenators. Our review describes novel epigenetic mechanisms that underpin myocardial remodeling, repair/ regeneration or senescence in order to support the development of most effective and reproducible rescue therapy of adult heart

Hydrogen sulfide in the mouse ductus arteriosus: a naturally occurring relaxant with potential EDHF function

We have previously reported that bradykinin relaxes the fetal ductus arteriosus via endothelium-derived hyperpolarizing factor (EDHF) when other naturally occurring relaxants (prostaglandin E(2), nitric oxide, carbon monoxide) are suppressed, but the identity of the agent could not be ascertained. Here, we have examined in the mouse whether hydrogen sulfide (H(2)S) is a relaxant of the ductus and, if so, whether it may also function as an EDHF. We found in the vessel transcripts for the H(2)S synthetic enzymes, cystathionine-\u3b3-lyase (CSE) and cystathionine-\u3b2-synthase (CBS), and the presence of these enzymes was confirmed by immunofluorescence microscopy. CSE and CBS were distributed across the vessel wall with the former prevailing in the intimal layer. Both enzymes occurred within the endoplasmic reticulum of endothelial and muscle cells, while only CSE was located also in the plasma membrane. The isolated ductus contracted to inhibitors of CSE (D,L-propargylglycine, PGG) and CBS (amino-oxyacetic acid, AOAA), and PGG contraction was attenuated by removal of the endothelium. EDHF-mediated bradykinin relaxation was curtailed by both PGG and AOAA, while the relaxation to sodium nitroprusside was not affected by either treatment. The H2S donor, sodium hydrogen sulfide (NaHS), was also a potent, concentration-dependent relaxant. We conclude that the ductus is endowed with a H(2)S system exerting a tonic relaxation. In addition, H(2)S, possibly via an overriding CSE source, qualifies as an EDHF. These findings introduce a novel vasoregulatory mechanism into the ductus, with implications for antenatal patency of the vessel and its transitional adjustments at birth

siRNA-mediated targeting of endothelial VWF prevents ET-1 upregulation in porcine aortic endothelial cells chronically exposed to angiotensin II

Objectives: The expression of endothelin (ET)-1, a potent vasoconstrictor and proinflammatory peptide, is increased in endothelial cells exposed to angiotensin II (Ang II). High levels of ET-1 progressively lead to endothelial dysfunction up to cardiovascular disorders. Since von Willebrand Factor (vWF) blockade improves endothelial function in coronary patients, we hypothesized that targeting endothelial vWF expression with small interference RNA (siRNA) prevents the Ang II-induced ET-1 upregulation. Materials and methods: see Results. Results: Nearly 65±2% silencing of vWF in porcine aortic endothelial cells (PAOECs) was achieved with selective siRNA (siRNA-vWF) without affecting cell viability and growth. At rest, vWF-knockdown PAOECs showed ET-1 levels similar to wild-type cells. Conversely, siRNA-vWF prevented the ET-1 upregulation after exposure to Ang II (100nM/24h), yet vWF levels were similar to unstressed cells. Even if siRNA-vWF significantly reduced eNOS expression, the levels of phospho-Ser1177-endothelial-nitric oxide synthase (eNOS)/eNOS ratio and nitric oxide in siRNA-vWF cells were similar to unstressed wildtype cells. Compared with wild-type cells, siRNA-vWF treatment significantly prevented the AngII-induced increase in NADPH oxidase activity and superoxide anion (O2-) levels, which mediate Ang II-induced ET-1 expression. Unraveling the mechanisms underlie vWF downregulation, the long-term treatment of cells with phorbol ester (PMA, 5nM/48h), which is a known activator of the NADPH-derived O2- production, did not increase O2- and ET-1 levels in vWF-knockdown cells. Conclusions: siRNA-based targeting of endothelial vWF prevented the Ang II-induced ET-1 upregulation through attenuation of O2- production due to inhibition of NADPH activity. Our findings reveal a hitherto unsuspected role of vWF in the prevention of Ang IIinduced endothelial dysfunction

EDHF function in the ductus arteriosus: evidence against involvement of epoxyeicosatrienoic acids and 12S-hydroxyeicosatetraenoic acid

We have previously shown (Ref. 2) that endothelium-derived hyperpolarizing factor (EDHF) becomes functional in the fetal ductus arteriosus on removal of nitric oxide and carbon monoxide. From this, it was proposed that EDHF originates from a cytochrome P-450 (CYP450)-catalyzed reaction being inhibited by the two agents. Here, we have examined in the mouse ductus whether EDHF can be identified as an arachidonic acid product of a CYP450 epoxygenase and allied pathways. We did not detect transcripts of the mouse CYP2C subfamily in vessel, while CYP2J subfamily transcripts were expressed with CYP2J6 and CYP2J9. These CYP2J hemoproteins were also detected in the ductus by immunofluorescence microscopy, being colocalized with the endoplasmic reticulum in both endothelial and muscle cells. Distinct CYP450 transcripts were also detected and were responsible for ω-hydroxylation (CYP4A31) and 12R-hydroxylation (CYP4B1). Mass spectrometric analysis showed formation of epoxyeicosatrienoic acids (EETs) in the intact ductus, with 11,12- and 14,15-EETs being more prominent than 5,6- and 8,9-EETs. However, their yield did not increase with nitric oxide/carbon monoxide suppression, nor did it abate with endothelium removal. No evidence was obtained for formation of 12R-hydroxyeicosatrienoic acid and ω-hydroxylation products. 2S-hydroxyeicosatetraenoic acid was instead detected, and, contrary to data implicating this compound as an alternative EDHF, its suppression with baicalein did not modify the EDHF-mediated relaxation to bradykinin. We conclude that none of the more common CYP450-linked arachidonic acid metabolites appears to qualify as EDHF in mouse ductus. We speculate that some novel eicosanoid or a totally unrelated compound requiring CYP450 for its synthesis accounts for EDHF in this vessel