Oxygen sensing in the ductus arteriosus-A unifying vision for two concepts.

Closure of the ductus arteriosus at birth is known to be related to the physiological rise in blood oxygen tension. Two main schemes have been proposed to explain the peculiar contraction of the ductus to oxygen, and their function is based on distinct sensor-effector complexes. Specifically, these schemes comprise a cytochrome P450 monooxygenase-endothelin complex or, alternatively, a mitochondrial redox mechanism-voltage gated potassium channel complex. However, it is not clear how these systems may relate to the closure process. Here, it is proposed that they operate jointly within a single functional entity, with their combined activation being required for a full-fledged contraction of the vessel to oxygen. This arrangement is thought to ensure a smooth transition from the powerful relaxant mechanism that is evident through the early postnatal period. Validation of this concept would not only settle a central question in the operation of the ductus but may also afford a broader perspective in any future translational research.

Potential markers of healing from near infrared spectroscopy imaging of venous leg ulcer. A randomized controlled clinical trial comparing conventional with hyperbaric oxygen treatment.

The aim of this study is to ascertain whether the simultaneous measurement of hemoglobin O2 saturation (StO2 ) and dimension of venous leg ulcers (VLU) by near infrared spectroscopy (NIRS) imaging can predict the healing course with protocols employing a conventional treatment alone or in combination with hyperbaric oxygen therapy (HBOT). NIRS 2D images of wound region were obtained in 81 patients with hard-to-heal VLU that had been assigned, in a randomized controlled clinical trial, to the following protocols: 30 HBOT sessions, adjunctive to the conventional therapy, either twice daily over 3 weeks (group A) or once daily over 6 weeks (group B), and conventional therapy without HBOT (group C). Seventy-three patients completed the study with a total of 511 NIRS images being analyzed. At the end of treatment, wound area was significantly smaller in all three groups. However, at the 3-week mark the wound area reduction tended to be less evident in group A than in the other groups. This trend continued up to the 6-week end-point when a significantly greater area reduction was found with group B (65.5%) and group C (56.8%) compared to group A (29.7%) (P < .01). Furthermore, a higher incidence of complete healing was noted with group B (20%) than with group A (4.5%) and group C (3.8%). When using a final wound reduction in excess of 40% to distinguish healing from nonhealing ulcers, it was found that only the former present NIRS StO2 values abating over the study period both at center and edge of lesions. In conclusion, NIRS analysis of StO2 and wound area can predict the healing course of VLU. Adjunctive HBOT significantly facilitates VLU healing compared to the conventional treatment alone. This positive action, however, becomes manifest only with a longer and less intensive treatment schedule.

Selective perfusion of coronary vasculature in preterm sheep: a methodological innovation undermined by unfavourable operation of the foramen ovale.

Antenatal cardiac intervention affords new prospects for hypoplastic left heart syndrome. Its success, however, may come not only from absence of impediments to blood flow but also from a sufficiently developed cardiac wall. Here, we examined the feasibility to perfuse selectively the fetal coronary circulation for treatment with growth promoting agents. Pregnant sheep (94-114 days gestation, term 145 days) were used. An aortic stop-flow procedure was developed for intracoronary access in the nonexposed fetus and human mesenchymal stem cells and their exosomes served as test agents. We found that aortic stop-flow ensures preferential distribution of fluorescent microspheres to the heart. However, intracoronary administration of stem cells or exosomes was detrimental, with fetal demise occurring around surgery or at variable intervals afterwards. Coincidentally, stop-flow caused by itself a marked rise of intraluminal pressure within the occluded aorta along with histological signs of coronary obstruction. We conclude that it is feasible to perfuse selectively the coronary circulation of the preterm fetus, but treatments are not compatible with survival of the animals. The cause for failure is found in the absence of hemodynamic compensation to stop-flow via a left-to-right shunt. This unexpected event is attributed to a largely membranous foramen ovale, characteristic of sheep, that collapses under pressure.

Differential proteome profile in ischemic heart disease: Prognostic value in chronic angina versus myocardial infarction. A proof of concept.

The initial clinical manifestation of ischemic heart disease (IHD) i.e. unheralded myocardial infarction (MI) versus chronic angina pectoris (AP) is statistically associated with adverse or mild disease progression respectively in the long-term follow-up. Here, we subjected AP and MI patients to blood proteomic analysis by Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS) in order to investigate putative new prognostic biomarkers of IHD manifestation. We found several differentially expressed peaks but four of them (4176, 4475, 14,158m/z and 8922m/z for AP and MI, respectively) were most reliable. Two of them were identified; 14,158m/z peak was the double-charged form of Apolipoprotein A-I and its vasoprotective action accords with prominence in AP. The 4176m/z peak was related to FAM83C protein, while neither the 4475m/z peak nor the MI-linked 8922m/z peak could be identified. We conclude that SELDI-TOF-MS analysis may yield a panel of molecular signals able to retrospectively classify patients according to their clinical and molecular features, exploitable for predicting the natural course of IHD.

Functional closure of the ductus arteriosus at birth: evidence against an intermediary role of angiotensin II.

The fetal ductus arteriosus (DA) closes postnatally first functionally and then structurally. Normal rise in blood oxygenation is regarded as a prime trigger, but closure may occur more slowly without this stimulus. Here, our aim was to assess the role of angiotensin II (Ang II) in functional closure of DA since its action may not be conditioned by oxygen. Experiments were performed with wild-type fetal and neonatal mice, using whole-body freezing technique to assess DA caliber in vivo. Transcripts for Ang II type 1 (AT1R) and type 2 (AT2R) receptors were also examined. We found that the AT1R antagonist olmesartan had no effect in the fetus, but delayed ductus closure in the neonate. However, this response was short-lived and disappeared upon concomitant treatment with the AT2R antagonist PD123319. Coincidentally, olmesartan promoted the Agtr2 transcript. We conclude that AT1R-based Ang II has no role in the functional closure of DA. Conversely, the compound may modulate this process through AT2R-mediated vasodilatation.

Therapeutic manipulation of the ductus arteriosus: current options and future prospects.

The ductus arteriosus is a large fetal vessel connecting the pulmonary artery with the aorta and allowing right ventricular blood to bypass the unexpanded lungs. At birth, with the start of lung ventilation and the attendant rise in blood oxygen tension, the ductus closes and the cardiovascular system acquires its final arrangement. However, in the prematurely born infant, this shunt may remain patent (patent ductus arteriosus--PDA) with adverse consequences on hemodynamic homeostasis. Conversely, there are cardiac malformations in which patency of the duct is required to maintain the pulmonary or systemic circulation prior to corrective surgery. Based on the notion that patency is an active process sustained primarily by prostaglandin (PG) E2, PDA is currently managed with synthesis inhibitors, indomethacin or ibuprofen, while any necessary persistence of the duct after birth is achieved with the infusion of PGE1. However, the former procedure presents a relatively high incidence of failures for the likely combination of the 2 events: the relaxing influence of the agents compensating for the loss of PGE2 and the immaturity of the oxygen­triggered contractile mechanism. On the other hand, PGE1 treatment loses some of its efficacy with time and may also be complicated by troublesome side effects. This article presents possible new approaches to therapy still based on the manipulation of the relaxing mechanism(s) responsible for duct patency. At the same time, however, the idea is put forward that the management of these sick infants may find its definitive solution only with tools being designed on the operation of the oxygen­sensing/effector system.

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.

Feto-placental vascular dysfunction as a prenatal determinant of adult cardiovascular disease.

Low birthweight is associated with increased rates of coronary heart disease, stroke, hypertension and non-insulin-dependent diabetes during adult life. This is thought to be the consequence of a 'programming', whereby a stimulus or insult at a critical, sensitive period of early life has permanent effects on structure, physiology and metabolism. Programming of the fetus may, hence, result from adaptations to a condition where placental nutrient supply fails to match fetal demand. Recently, compensatory feto-placental up-regulation of the nitric oxide system during fetal growth restriction (FGR) was shown. Particularly, restricted hypoxic fetuses present an elevation of nitrites and a reduction of asymmetric dimethylarginine. S-nitrosohemoglobin is consumed under hypoxic conditions. These events are followed by nitric oxide pathway down-regulation postnatally, increasing susceptibility to cardiovascular disorders later in life. The relative hyperoxia would favor any such occurrence through depletion of tetrahydrobiopterin secondary to oxygen radical formation. This concept may lead to new therapeutic strategies, based on tetrahydrobiopterin supplementation, free-radical scavenging, L-arginine administration and/or inhaled NO therapy in FGR hypoxic newborns, to improve their postnatal adaptation and to reduce the risk of metabolic pathologies in adult age.

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.

Compensatory feto-placental upregulation of the nitric oxide system during fetal growth restriction.

BACKGROUND: Fetal Growth Restriction is often associated with a feto-placental vascular dysfunction conceivably involving endothelial cells. Our study aimed to verify this pathogenic role for feto-placental endothelial cells and, coincidentally, demonstrate any abnormality in the nitric oxide system. METHODS: Prenatal assessment of feto-placental vascular function was combined with measurement of nitric oxide (in the form of S-nitrosohemoglobin) and its nitrite byproduct, and of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine. Umbilical vein endothelial cells were also harvested to determine their gene profile. The study comprised term pregnancies with normal (n = 40) or small-for-gestational-age (n = 20) newborns, small-for-gestational-age preterm pregnancies (n = 15), and bi-chorial, bi-amniotic twin pregnancies with discordant fetal growth (n = 12). RESULTS: Umbilical blood nitrite (p<0.001) and S-nitrosohemoglobin (p = 0.02) rose with fetal growth restriction while asymmetric dimethylarginine decreased (p = 0.003). Nitrite rise coincided with an abnormal Doppler profile from umbilical arteries. Fetal growth restriction umbilical vein endothelial cells produced more nitrite and also exhibited reciprocal changes in vasodilator (upwards) and vasoconstrictor (downwards) transcripts. Elevation in blood nitrite and S-nitrosohemoglobin persisted postnatally in the fetal growth restriction offspring. CONCLUSION: Fetal growth restriction is typified by increased nitric oxide production during pregnancy and after birth. This response is viewed as an adaptative event to sustain placental blood flow. However, its occurrence may modify the endothelial phenotype and may ultimately represent an element of risk for cardiovascular disease in adult life.

Mechanisms for ductus arteriosus closure.

Closure of the ductus arteriosus at birth is a complex phenomenon being conditioned by antenatal events and progressing in preprogrammed steps. Functional at first, narrowing of the vessel is determined by 2 overlapping processes--removal of the prostaglandin E(2)-based relaxation sustaining prenatal patency and activation of a constrictor mechanism by the natural rise in blood oxygen tension. Two schemes have been proposed for oxygen action--one involving a cytochrome P450 hemoprotein (sensor)/endothelin-1 (effector) complex and the other a set of voltage-gated K(+) channels. These proposals, however, are not mutually exclusive. Structural closure follows the constriction through a remodeling process initiated antenatally with the development of intimal cushions and completed postnatally by a host of humoral and mechanical stimuli. Research in this area has already provided clinical applications. Nevertheless, management of premature infants with persistent ductus remains troublesome and calls for an alternative approach to the prostaglandin E(2) inhibitors now in use. Studies in progress on the oxygen-sensing system may lead to a definitive solution for this problem.

Arachidonic acid epoxygenase and 12(S)-lipoxygenase: evidence of their concerted involvement in ductus arteriosus constriction to oxygen.

Oxygen promotes closure of the ductus arteriosus at birth. We have previously presented a scheme for oxygen action with a cytochrome P450 (CYP450) hemoprotein and endothelin-1 (ET-1) being, respectively, sensor and effector, and a hypothetical monooxygenase product serving as a coupling link. We have also found in the vessel arachidonic acid (AA) 12(S)-lipoxygenase (12-lipoxygenase) undergoing upregulation at birth. Here, we examined the feasibility of a sensor-to-effector messenger originating from AA monooxygenase and 12-lipoxygenase pathways. The epoxygenase inhibitor, N-methylsulfonyl-6-(2-)hexanamide, suppressed the tonic contraction of ductus to oxygen. A similar effect was obtained with 12-lipoxygenase inhibitors baicalein and PD 146176. By contrast, none of the inhibitors modified the endothelin-1 contraction. Furthermore, an AA ω-hydroxylation product, 20-hydroxyeicosatetraenoic acid (20-HETE), reportedly responsible for oxygen contraction in the systemic microvasculature, had no such effect on the ductus. We conclude that AA epoxygenase and 12-lipoxygenase jointly produce a hitherto uncharacterized compound acting as oxygen messenger in the ductus.

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.

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.

Antenatal corrective cardiac surgery: An emerging area for technological innovation.

The possibility of intervening in utero on certain cardiac malformations with the intent to prevent secondary major alterations in structure and function is becoming a reality. Central to progress in this area is the development of instrumentation specifically designed for minimally invasive cardiac surgery in the fetus. The present review introduces a novel set of devices for interventional cardiology, based on current knowledge and prior experience, and highlights their prospective application. In meeting this objective, particular importance is assigned to the synergic contribution of diverse disciplines, both medical and nonmedical.

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.