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 E2, nitric oxide, and carbon monoxide) are suppressed, but the identity of the agent could not be ascertained. Here, we have examined in the mouse whether hydrogen sulfide (H2S) 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 H2S synthetic enzymes, cystathionine-γ-lyase (CSE) and cystathionine-ß-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, whereas only CSE was located also in the plasma membrane. The isolated ductus contracted to inhibitors of CSE (d,l-propargylglycine, PPG) and CBS (amino-oxyacetic acid), and PPG contraction was attenuated by removal of the endothelium. EDHF-mediated bradykinin relaxation was curtailed by both PPG and amino-oxyacetic acid, whereas 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 H2S system exerting a tonic relaxation. In addition, H2S, 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.

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.

Cytochrome P-450 3A13 and endothelin jointly mediate ductus arteriosus constriction to oxygen in mice.

The fetal ductus arteriosus (DA) contracts to oxygen, and this feature, maturing through gestation, is considered important for its closure at birth. We have previously obtained evidence of the involvement of cytochrome P-450, possibly of the 3A subfamily (CYP3A), in oxygen sensing and have also identified endothelin (ET)-1 as the attendant effector for the contraction. Here, we examined comparatively wild-type (WT) and CYP3A-null (Cyp3a(-/-)) mice for direct validation of this concept. We found that the CYP3A subfamily is represented only by CYP3A13 in the WT DA. CYP3A13 was also detected in the DA by immunofluorescence microscopy, being primarily colocalized with the endoplasmic reticulum in both endothelial and muscle cells. However, a distinct signal was also evident in the plasma membrane. Isolated DAs from term WT animals developed a sustained contraction to oxygen with transient contractions superimposed. Conversely, no tonic response occurred in Cyp3a(-/-) DAs, whereas the phasic response persisted unabated. Oxygen did not contract the preterm WT DA but caused a full-fledged contraction after retinoic acid (RA) treatment. RA also promoted an oxygen contraction in the Cyp3a(-/-) DA. However, responses of RA-treated WT and Cyp3a(-/-) mice differed in that only the former abated with ET-1 suppression. This implies the existence of an alternative target for RA responsible for the oxygen-induced contraction in the absence of CYP3A13. In vivo, the DA was constricted in WT and Cyp3a(-/-) newborns, although with a tendency to be less narrowed in the mutant. We conclude that oxygen acts primarily through the complex CYP3A13 (sensor)/ET-1 (effector) and, in an accessory way, directly onto ET-1. However, even in the absence of CYP3A13, the DA may close postnatally thanks to the contribution of ET-1 and the likely involvement of compensating mechanism(s) identifiable with an alternative oxygen-sensing system and/or the withdrawal of relaxing influence(s) operating prenatally.

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 omega-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 omega-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.

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.

Detection of mtDNA with 4977 bp deletion in blood cells and atherosclerotic lesions of patients with coronary artery disease.

Recent evidence suggests that somatic mutations in nuclear and mitochondrial DNA accumulated during aging, may significantly contribute to the pathogenesis of chronic-degenerative illness such as coronary artery disease (CAD). Mitochondrial DNA with 4977 bp deletion mutation (mtDNA4977) is a common type of mtDNA alteration in humans. However, little attempt has been made to detect the presence of mtDNA4977 deletion in cells and tissues of cardiovascular patients. This study investigated the presence of mtDNA4977 in blood samples of 65 cardiovascular patients and 23 atherosclerotic plaques of human coronaries with severe atherosclerosis. Moreover, the presence of the deletion has been investigated in blood cells from 22 healthy age-matched subjects. The detection of mtDNA4977 has been performed by using a nested polymerase chain reaction (PCR) protocol and normalized to wild-type mtDNA. A significant higher incidence of mtDNA4977 was observed in CAD patients with respect to healthy subjects (26.2% versus 4.5%; P=0.03). Furthermore, the relative amount of the deletion was significantly higher in the patients compared to the control group (P=0.02). The mtDNA4977 was detected in 17 of the 65 patients blood samples (26.2%) and deletion levels ranged from 0.18 to 0.46% of the total mtDNA (mean: 0.34+/-0.02%). For what concerns atherosclerotic lesions, 5 patients (21.7%) showed the deletion ranging from 0.13 to 0.45% of the total mtDNA (mean: 0.35+/-0.06%). In both samples from patients, the incidence and the relative amount of mtDNA4977 was not significantly influenced by atherogenic risk factors and clinical parameters. The obtained results may suggest that the increase of oxidative stress in cardiovascular disease may be responsible for the accumulation of mtDNA damage in coronary artery disease patients.

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.

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.

Endothelial function and carotid intima-media thickness in young healthy subjects among endothelial nitric oxide synthase Glu298-->Asp and T-786-->C polymorphisms.

BACKGROUND AND PURPOSE: To assess the role of the endothelial nitric oxide synthase (eNOS) gene variants as risk factors for early atherosclerosis, we sought to investigate whether two polymorphisms located in the exon 7 (Glu298-->Asp) and in the promoter region (T-786-->C) of the eNOS gene were associated with functional changes in the endothelium and carotid intima-media thickness (IMT). METHODS: Endothelium-dependent flow-mediated brachial artery dilation (FMD), endothelium-independent dilation response to glyceryl trinitrate (GTN), and carotid IMT were assessed by high-resolution ultrasound in 118 healthy young nonsmoker subjects (30.1+/-0.5 years) genotyped for the eNOS Glu298-->Asp and T-786-->C polymorphisms. RESULTS: Carotid IMT was inversely related to FMD by univariate analysis (r=-0.28, P=0.002) and after adjustment for possible confounders in all the subjects (P<0.01). Asp homozygotes had a significantly lower FMD than Glu carriers (Glu/Glu: 15.0%+/-1.0%, Glu/Asp: 13.3%+/-0.7%, Asp/Asp: 9.6%+/-1.6%; P=0.005), whereas FMD was unaffected by the T-786-->C variant. Neither the Glu298-->Asp nor the T-786-->C polymorphisms influenced the GTN-mediated dilation. With respect to Glu carriers, Asp/Asp genotype displayed a significantly greater carotid IMT (Glu/Glu: 0.37+/-0.01 mm, Glu/Asp: 0.35+/-0.01 mm, Asp/Asp: 0.45+/-0.03 mm; P=0.0002) and significant correlations between carotid IMT and FMD (r=-0.48, P=0.04) and between carotid IMT and resting brachial artery diameter (r=0.70, P=0.001). No difference in IMT was found across the T-786-->C genotypes. By multivariate regression analysis, Asp/Asp genotype was the only significant and independent predictor of flow-mediated brachial artery dilation (FMD) (P=0.04) and carotid intima-media thickness (IMT) (P=0.006). CONCLUSIONS: The eNOS Glu298-->Asp polymorphism may be related to early atherogenesis.

Weight is an independent predictor of vascular injury in healthy volunteers with aspartate allele.

BACKGROUND: Endothelial dysfunction and carotid intima-media thickeness (IMT) are currently considered key early events in atherogenesis and markers of arterial damage. We investigated whether endothelial nitric oxide synthase (eNOS) glutamate (Glu)298-aspartate (Asp) polymorphism may influence the vascular response to weight, as measured by BMI, in young, healthy individuals. METHODS: One hundred young (30.6 ±â€Š5.9 years) healthy individuals, without concomitant traditional cardiovascular risk factors took part in the study. Brachial artery endothelial function was assessed by vascular response to reactive hyperemia [flow-mediated dilation (FMD) and sublingual nitroglycerin (GTN)-mediated dilation] using high-resolution ultrasound. Carotid IMT was also measured. RESULTS: Participants were grouped as Glu-homozygotes (n = 38) and Asp-carriers (n = 62). On univariate analysis, a higher response to GTN was associated with lower brachial baseline diameter (P < 0.001) and increasing value of high-density lipoprotein cholesterol (P = 0.04) in Asp-carriers, but not in Glu-homozygotes. Higher FMD correlated with lower brachial baseline diameter (P < 0.001), BMI (P = 0.03) and SBP (P = 0.03) in the Asp-carriers, but not in Glu-homozygotes. Higher IMT showed a similar Asp-genotype-dependent association with higher BMI (P = 0.001), SBP (P = 0.006) and DBP (P = 0.001). In individuals with Asp-alleles, the multivariate analysis showed that BMI was the only independent predictor of IMT. CONCLUSION: Weight is independently associated with impaired arterial structure in healthy and genetically predisposed young individuals. The allelic variation (Asp298) of the eNOS gene polymorphism makes individuals vulnerable to the impact of weight on the development of atherosclerosis.

Dual, constrictor-to-dilator, response of the mouse ductus arteriosus to the microsomal prostaglandin E synthase-1 inhibitor, 2-(6-chloro-1H-phenanthro[9,10d]imidazole- 2-yl)isophthalonitrile.

BACKGROUND: Microsomal prostaglandin E synthase-1 (mPGES1) is critical for prostaglandin E(2) formation in ductus arteriosus (DA) and, accordingly, in its patency. We previously reported that mPGES1 deletion, unlike cyclo-oxygenase (COX) suppression, is not followed by upregulation of relaxant nitric oxide (NO). Consequently, we proposed that a mPGES1 inhibitor may be better than currently used COX inhibitors in managing premature infants with persistent DA (PDA). OBJECTIVE: To assess the effect of the mPGES1 inhibitor, 2-(6-chloro-1H-phenanthro[9,10d]imidazole-2-yl)isophthalonitrile (MF63) on DA ex vivo and in vivo (p.o. to the mother). METHODS: Experiments were carried out with mice bearing human mPGES1. We utilized isolated, wire-mounted DA for isometric recording and a whole-body freezing technique to assess the DA caliber as it occurs in vivo. RESULTS: MF63 (10 µM) contracted the isolated DA. DA constriction was also seen in vivo after a single 10-mg kg(-1) dose. Conversely, a 30-mg kg(-1) dose gave inconsistent results, combining constriction with no effect. DA dilatation followed instead a repeated lower dose (twice daily for 3 days), and postnatal closure of the vessel was also delayed. Chronic pretreatment had no effect on endothelial NO synthase mRNA expression in fetal DA, nor did it modify the contraction to NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (100 µM). CONCLUSIONS: MF63 has a dual action on DA, the constriction being associated with accessory dilatation. The latter effect should be explained before considering further a mPGES1 inhibitor for management of PDA.

ET-1 Lys198Asn and ET(A) receptor H323H polymorphisms in heart failure. A case-control study.

BACKGROUND: The endothelin (ET) system plays a central role in the control of myocardial function and its pathophysiology. The aim of the present study was to explore whether genetic variations of ET-1 (G/T substitution that predicts an Lys/Asn change at codon 198) and its receptor ET(A) (T/C in exon 6, H323H) could predispose carriers to heart failure (HF). METHODS: Genotyping at these two loci was done in 122 patients with HF [echocardiographic left ventricular ejection fraction (LVEF) < or =40%] and 216 age-matched subjects without HF. Causes of HF included ischemic (n = 96) and idiopathic cardiomyopathies (n = 26). RESULTS: The ET-1 Lys198Asn was significantly associated with the occurrence of HF (p = 0.005). The risk of HF was independently increased among Asn/Asn in comparison to Lys carriers (OR = 3.2, p = 0.03). Moreover, homozygous carriers of both ET-1 and ET(A) variants showed a marked increase in the risk of HF (adjusted OR = 8.6, p = 0.005), displayed significantly lower LVEF (p = 0.002) and higher left ventricular end-diastolic (p = 0.03) and end-systolic diameters (p = 0.04; for Asn/Asn and TT vs. Lys and C carriers of the ET-1 and ET(A )polymorphisms, respectively). Furthermore, the extent of coronary artery disease (r = -0.62, p < 0.0001) and the Asn/Asn and TT double genotype (r = -0.30, p = 0.0001) were the only significant and independent predictors of LVEF by multivariate analysis. CONCLUSIONS: The ET-1 Lys198Asn and ET(A) receptor H323H polymorphisms seem to act synergistically to increase the risk of HF.