The renin-angiotensin system plays a key role in the initiation and maintenance of elevated blood pressure associated with altered intrauterine milieu. The current studies were undertaken to verify whether vascular response to ANG II is increased in adult offspring of low-protein fed dams (LP) compared with control (CTRL) and if so, to examine underlying mechanism(s). ANG II-induced contraction of carotid rings was increased in LP (Emax, the maximum asymptote of the curve, relative to maximal response to KCl 80 mM: 230 ± 3% LP vs. 201 ± 2% CTRL, P < 0.05). In both groups, contraction to ANG II was mediated solely by AT1R. Responses to thromboxane A2 analog U-46619 and to KCl 80 mM under step increases in tension were similar between groups. Endothelium depletion enhanced contraction to ANG II in both groups, more so in LP. Blockade of endothelin formation had no effect on response to ANG II, and ANG-(1–7) did not elicit vasomotor response in either group. Superoxide dismutase (SOD) analog Tempol normalized LP without modifying CTRL response to ANG II. Basal levels of superoxide (aortic segments, lucigenin-enhanced chemiluminescence and fluorescent dye hydroethidine) were higher in LP. ANG II further increased superoxide production in LP only, and this was inhibited by coincubation with diphenylene iodonium or apocynin (inhibitor of NADPH oxidase complex). AT1R expression in carotid arteries was increased in LP, whereas SOD expression was unchanged. In conclusion, vasoconstriction to ANG II is exaggerated in this model of developmental programming of hypertension, secondary to enhanced vascular production of superoxide anion by NADPH oxidase with concomitant increase of AT1R expression.
- oxidative stress
- isolated vessels
epidemiological studies have revealed that the risk of cardiovascular diseases such as hypertension, stroke, and coronary heart disease in later life seem inversely related to birth weight (23) and that this relation is independent of genetic factors and life style (1, 3). These observations led to the postulate that perturbations (of nutrition, for example) at a critical period of early development could lead to permanent alterations in the programming of the developing cardiovascular structures or functions (2). Animal studies support the concept of developmental programming of hypertension (17, 19, 25, 46).
The renin-angiotensin system (RAS) has been shown to play a key role in the initiation and maintenance of elevated blood pressure associated with altered intrauterine milieu. Blockade of ANG II formation or of ANG II AT1 receptor (AT1R) subtype during the first weeks of life prevents later elevation of blood pressure; this permanent effect is not observed when adult animals are treated (41, 42). We and others have demonstrated that in adult offspring of dams fed a low-protein (LP) diet during gestation, plasma renin activity is increased, elevated blood pressure is normalized by angiotensin-converting enzyme inhibitor (ACEi), and pressor response to infusion of ANG II is increased (20, 26, 33, 34). Circulating ANG II increases blood pressure through peripheral and central mechanisms. We have previously demonstrated increased expression of ANG II AT1R in brain cardiovascular regulating areas of adult male LP offspring and normalization of their blood pressure with intracerebroventricular injection of the ACEi enalaprilat or of the AT1R antagonist losartan (33).
The current studies were undertaken to verify whether peripheral vascular response to ANG II is also increased in adult male LP offspring compared with control male offspring (CTRL) and to explore potential mechanisms underlying the exaggerated vasoconstriction to ANG II. For this purpose, vasomotor responses of carotid arteries rings (organ chambers) were studied. Increased vasomotor response to ANG II could be observed in the presence of vascular remodeling and/or increase in the expression of ANG II AT1R subtype, decrease in the expression of ANG II AT2R subtype, which favors vasodilatation, or changes in ANG II-mediated signal transduction at or beyond the level of the cell membrane receptor, as it has been described in other forms of chronic hypertension (45). Also, increased vasoconstriction can be encountered in the presence of defective vasodilatation, such as decreased endothelium (nitric oxide)-mediated vasodilatation classically reported in many human cases or animal models of chronic hypertension. To explore these potential mechanisms, the following studies were undertaken: As we and others reported unchanged histological measurements of arteries (media, lumen) from LP offspring (6, 34), we verified whether tensional force capacity was also unaffected by antenatal diet exposure. To sort out the role of AT1R vs. AT2R in vasomotor response to ANG II, specific antagonists were used; however, expression of AT2R has not been reported in nonlesioned carotid arteries. Activation of AT1R can lead to enhanced formation of endothelin, as well as superoxide, mostly through activation of NADPH oxidase, both of which can enhance vasoconstriction (40, 44). Alternatively, ANG II can lead to vasodilation through metabolite ANG-(1–7) and through stimulation of NO production (5, 9). The latter potential mechanisms were studied using specific blockers. Defective endothelium-mediated vasodilation was studied in vessel rings denuded of their endothelium. As our results support our primary hypothesis and reveal that the exaggerated constriction to ANG II is normalized by superoxide dismutase (SOD) analog Tempol, the vascular production of superoxide anion was also evaluated.
MATERIALS AND METHODS
Animals were used according to a protocol approved by the Animal Care Committee of Sainte-Justine Hospital in accordance with the principles of the Guide for the Care and Use of Experimental Animals of the Canadian Council on Animal Care. Virgin Wistar rats (initial weight 225–250 g), which were mated overnight and on the day of conception (determined by the presence of a vaginal plug), were allocated to feed ad libitum on a diet containing either 18% [control group (CTRL), n = 12] or 9% (LP group, n = 11) casein (20). All diets contained 5 g/kg methionine to avoid sulfur deficiency and were made isocaloric with starch and sucrose supplement. All dams were weighed daily and had free access to food and water. Within 12 h of delivery, dams were returned to regular chow. Pups were weaned at 4 wk of age to regular chow, and males were studied at 9–12 wk of age. Unless specified otherwise, one animal per litter was used for the different studies.
Ex vivo vascular reactivity studies.
Freshly excised carotid arteries from anesthetized [intraperitoneal ketamine (65 mg/kg) and xylazine (7 mg/kg)] offspring were placed in ice cold modified Krebs bicarbonate solution (KBS) of the following composition (in mM): 118 NaCl, 4.7 KCl, 25 NaHCO3, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, and 11 dextrose. They were cleaned of adherent connective tissue and precisely cut into rings of same length (4 mm). Four to eight rings from one rat were used for one experiment. The n presented with each figure represents the number of animals studied. In a separate series of experiments, endothelium was removed in half of the rings by gently rubbing the internal lumen with a blunt needle (20 gauge; Becton Dickinson, Franklin Lakes, NJ). Rings were suspended horizontally between two stainless-steel wires in organ chambers that contained 20 ml of KBS maintained at 37°C and aerated continuously with 95% oxygen and 5% carbon dioxide. The tension of the preparations was recorded with a linear force transducer on a computerized data acquisition system (Kent Scientific, Litchfield, CT). The rings were progressively stretched to a preload tension of 19.0 mN and allowed to equilibrate for 30 min with frequent washing and tension adjustments. After stabilization, rings were repeatedly exposed to KCl (80 mM) to test their viability and to determine a standard contractile response for each of them. When response to KCl was stable, endothelial integrity was then assessed in all experiments by a characteristic relaxation response to carbachol (100 μM) after contraction evoked by phenylephrine (1 μM). Rings were then allowed to recover for 60 min, after which cumulative concentration-response curves were generated with ANG II (1 pM to 1 μM) in the presence and absence (half the rings tested each condition for each experiment) of PD 123319 (0.1 μM; ANG II AT2R antagonist) or losartan (1 μM; ANG II AT1R antagonist), phosphoramidon [1 μM, neutral endopeptidase 24.11 and endothelin converting enzyme inhibitor], Tempol (1 mM, SOD analog). All drugs were added to the bath 30 min before cumulative concentration-response curves. Cumulative concentration-response curves were also generated with the thromboxane A2 mimetic U46619 (1 pM to 1 μM) and with ANG-(1–7), (1 pM to 1 μM). For the determination of ANG-(1–7) vasorelaxant responses, rings were precontracted with U-46619 (0.3 μM) added to the organ chamber 15 min before. For the experiments in which the endothelium had been mechanically removed, absence of vasorelaxation to carbachol after normal vasoconstriction to KCl and phenylephrine was verified before the generation of the concentration-response curve to ANG II. To verify whether antenatal diet exposure was associated with changes in tensional force capacity, we also studied vasoconstriction to KCl (80 mM) under different tension applied to the carotid artery rings (48).
Evaluation of Vascular Production of Superoxide Anion by Chemiluminescence
Vascular superoxide production was estimated using lucigenin-enhanced chemiluminescence, as described (4, 24). Briefly, in an additional set of experiments, aortas were removed from anesthetized rats and cut into segments. Aortic rings (5 mm) were placed in polypropylene tubes each containing 0.5 ml of Krebs and lucigenin (5 μM). Chemiluminescence counts (expressed in relative light units) were measured (LS 6500 Multipurpose Scintillation Counter; Beckman Coulter) during 15 min (one reading/min) after preincubation for 30 min with Krebs solution, Krebs solution + ANG II (1 μM), Krebs solution + ANG II (1 μM) + diphenylene iodonium (DPI) (100 μM, inhibitor of flavin-containing enzyme) or Krebs solution + ANG II (1 μM) + apocynin (1 mM, inhibitor of the assembly of the NADPH oxidase complex). Background counts (Krebs solution alone) were subtracted from the counts obtained with vascular rings. Dry weight was obtained for each ring for normalization of activity.
Evaluation of Vascular Production of Superoxide Anion by Hydroethidine
Superoxide levels were also measured by the oxidative fluorescent dye hydroethidine, as described previously (27). Cells are permeable to hydroethidine and, in the presence of superoxide anion, hydroethidine is oxidized to fluorescent ethidium bromide, in which form, it is trapped by intercalation with DNA. Briefly, unfixed frozen aorta segments were cut into 12-μm-thick sections with a cryostat (Microm Cryostat, Waldorf, Germany) at −20°C and thaw-mounted on microscope slides (Superfrost, VWR Scientific, Pittsburgh, PA). Hydroethidine (2 μM) was applied to each tissue section and coverslipped. Then, slides were incubated in a light-protected humidified chamber at 37°C for 30 min. Images were obtained with a laser scanning confocal microscope (LSM 510 laser scanning microscope; Zeiss) equipped with a argon laser. Fluorescence was detected with a 514-nm long-pass filter. The LP and CTRL offspring were processed and imaged in parallel.
Western Blot Analysis
Western blot analysis of ANG II AT1 receptor subtype and of SOD enzyme was performed on carotid arteries of 9- to 12-wk-old male (LP: n = 4 from three litters and control: n = 5 animals from three litters). Frozen arterial tissues were disrupted using mortar and pestle in liquid nitrogen. Proteins were extracted with RIPA buffer supplemented with Nonidet P-40 (1%), Na3VO4 (1 mM), NaF (1 mM), EDTA (1 mM), and 1 × solution of cocktail protease inhibitors (Roche). Samples were sonicated and centrifuged, and the supernatant was recovered for determination of protein content by Bradford assay using BSA as a standard. For each experiment, tissues were pooled and blots were performed on equal amounts (30 μg) of total crude extract of protein. Polyclonal anti-β actin (Santa Cruz Biotechnology, Santa Cruz, CA) was used as internal control. Equal loading of proteins for SDS-PAGE was also verified by Ponceau staining. The antibodies used were the polyclonal anti-ANG II AT1 receptor subtype (Santa Cruz Biotechnology) and polyclonal anti-Cu/Zn SOD (Calbiochem, San Diego, CA).
The following agents were purchased: ketamine (Ayerst, Montreal, QC, Canada); xylazine (Bayer); ANG II, ANG-(1–7), apocynin, phosphoramidon, carbachol, phosphate buffer, EDTA, DPI, BSA, PD-123319 and U-46619 (Sigma, St Louis, MO); Tempol (Fluka Chemika, Buchs, Switzerland), losartan was a gift of Merck Frosst Canada and Du Pont (Kirkland, QC).
Cumulative concentration-response curves were analyzed by computer fitting to a four-parameter sigmoid curve using the Prism 3 program (GraphPad, San Diego, CA) to evaluate the half-maximal effective concentration (EC50) and Emax, the maximum asymptote of the curve. For the comparison of superoxide levels detected by chemiluminescence under control conditions vs. in the presence of ANG II ± DPI or apocynin, analysis was performed using ANOVA. Comparison of superoxide levels between groups was performed using unpaired Student's t-test. All values are expressed as means ± SE. A P value < 0.05 was considered significant.
Effect of Antenatal Diet Exposure on Ex Vivo Vasomotor Responses of Adult Male Offspring
The maximal constriction generated by ANG II (Emax) was significantly increased in LP compared with control offspring; EC50 value was not different between groups (Fig. 1A). Administration of losartan nearly totally inhibited the vasoconstriction to ANG II, whereas administration of PD-123319 was without effect in both groups (Fig. 1, B and C).
The vasoconstriction in response to KCl at different tension was not different between groups, indicating that the tensional force was not different between the groups (Fig. 2A). Vasoconstriction in response to U-46619 (Fig. 2B) was not altered by the different antenatal diet exposure.
Vasomotor response to ANG II was significantly enhanced after removal of the endothelium in both groups. Percent increase in Emax for LP offspring rings in absence vs. in the presence of endothelium was significantly more than for control offspring rings (Fig. 3, A and B).
Phosphoramidon, which blocks both endothelin-converting enzyme and neutral endopeptidase 24.11, which converts ANG I to ANG-(1–7), did not modify cumulative response curves to ANG II in either group (Fig. 4, A and B).
As ANG II can be metabolized by angiotensin-converting enzyme 2 to vasodilator ANG-(1–7), its potential role in modulating vasoconstriction to ANG II was examined. Cumulative doses of ANG-(1–7) elicited no vasomotor effect in either group. Integrity of the vasorelaxant properties of the rings was verified at the end of each experiment with carbachol (100 μM) (Fig. 4, C and D).
Effect of Tempol on Ex Vivo Vasomotor Response to ANG II
In the presence of Tempol, maximal vasoconstriction to ANG II of LP offspring ring was significantly decreased to values similar to control offspring. Tempol did not modify vasomotor response to ANG II of control offspring (Fig. 5, A and B).
Effect of Antenatal Diet Exposure on Vascular Production of Reactive Oxygen Species
Superoxide production in the aortic artery was measured using lucigenin-enhanced chemiluminescence (Fig. 6A). LP offspring basal levels of superoxide were significantly higher than control offspring. ANG II further increased significantly superoxide generation in LP offspring only; coincubation of the aortic segments with DPI or apocynin prevented the increased in superoxide generation observed in the presence of ANG II. In the control group, we did not observe differences in the levels of superoxide between baseline conditions and in the presence of ANG II ± DPI or apocynin.
Superoxide production evaluated by the oxidative fluorescent dye hydroethidine was also markedly increased in LP aorta and localized in vascular smooth muscle cells (Fig. 6, B and C).
Western Blot Analysis
AT1R expression in carotid arteries was significantly increased in LP, whereas SOD expression was not different between groups (Fig. 7).
In the current studies, we demonstrate exaggerated vascular (carotid arteries) response to ANG II in adult male offspring of dams fed a LP diet during gestation. This enhanced vascular response to ANG II seems specific, as a response to another vasoconstrictor U-44619, analog of thromboxane A2, was found unaltered in LP compared with control offspring. This vasomotor response is mediated by ANG II AT1R subtype, which expression is increased in LP offspring carotid arteries, and by the production of superoxide anions. We found no difference between groups in the carotid arteries expression of SOD. We show that production of superoxide anions is significantly increased in baseline conditions and in response to ANG II in LP offspring aortic wall and that this increased production of superoxide anion is mediated by NADPH oxidase. Our results also show that the exaggerated carotid arteries vasoconstriction to ANG II is not caused by increase in tensional force capacity of the vessel, by a defect in endothelium-released relaxing factor, or by secondary release of endothelin. Finally, the current results show that in both diet groups, vasomotor response to ANG II is not mediated by either AT2R or the ANG-(1–7) Mas receptor.
Many studies have demonstrated the key role of the RAS in the initiation and maintenance of hypertension associated with intrauterine protein restriction. In adult LP offspring, plasma renin activity is increased, and blood pressure is normalized by ACEi (20, 33, 34). The activity of ACE is elevated in LP offspring (although ANG II levels are not consistently found increased) (20, 21), which could suggest increased sensitivity of LP offspring to circulating ANG II. Supporting this are reports of increased blood pressure response to intravenously infused ANG II in LP adult offspring (26, 34), and the current data examining carotid arteries confirm this is the case.
Vascular hyperresponsiveness to ANG II, more markedly so than to other vasoconstrictors, has been reported in human and experimental animal models of hypertension (45). In developmentally programmed hypertension, other studies have also found no modification in Emax response to U-46619, as well as to phenylephrine in pial microvessels and mesenteric arteries (6, 16).
Increased vasomotor response to ANG II can be observed in the presence of vascular remodeling and/or increase in the expression of ANG II AT1R subtype; decrease in the expression of ANG II AT2R subtype, which favors vasodilatation; or changes in ANG II-mediated signal transduction (45). Alternatively, increased vasoconstriction can be encountered in the presence of defective vasodilatation.
Increased tensional force capacity seems absent in adult LP offspring. Vasomotor response to another potent constrictor U-46619 is not different between LP and control offspring. Constriction response curves of both groups to KCl under increasing tensions applied to the rings were nearly identical. These functional data are supported by previous reports by us and others showing no difference between groups in lumen diameter, media cross-sectional area, media thickness, and media-to-lumen ratio of the carotid and mesenteric arteries (6, 34).
We identified the AT1R as the sole receptor implicated in the carotid artery vasoconstriction response to ANG II for the two groups. Losartan nearly completely inhibited the vasoconstriction to ANG II, whereas PD-123319 did not modify the response to ANG II, suggesting that AT2R is not or functionally weakly expressed in rat carotid arteries (30, 32). However, we cannot exclude a role for AT2R in the resulting hypertension or in the ANG II in vivo pressor response in this model of programmed hypertension. Indeed, although not in vessels, renal AT2R mRNA expression is decreased in 4-wk-old female (but not male) LP offspring (26).
Carotid expression of AT1R is increased in LP offspring. On the basis of the Western blot analysis performed on whole vessels and the vasomotor response elicited by ANG II in the absence of endothelium, we conclude that AT1R is present on the vascular smooth muscle cells. AT1R had previously been studied in 4-wk-old male LP kidneys, in which protein expression (Western and binding analyses) was found increased (37, 45a), but mRNA expression remained unchanged (26). In another model of fetal programming of hypertension associated with a 50% global nutrient restriction of the dam during gestation, AT1R mRNA expression is also reported unchanged in mesenteric arteries of adult offspring (10). The underlying factors leading to enhanced AT1R vascular expression in LP offspring are unknown. One of the most potent elements affecting ANG II receptor expression is ANG II itself. In vitro and in vivo, ANG II decreases vascular smooth muscle cell AT1R expression (13, 22). Even though ACE and plasma renin activities are increased in adult LP offspring, circulating ANG II has been reported as unchanged or inconsistently increased in LP adult offspring (18). However, renal tissue ANG II is decreased in newborn (47); it could therefore be postulated that this decrease in neonatal ANG II, if it applies also to circulating ANG II, could permanently program vascular AT1R expression. The fact that others have reported unchanged mRNA allows one to consider posttranscriptional and/or posttranslational changes in the regulation of AT1R expression in LP offspring. Factors leading to enhanced expression of AT1R could also comprise corticosteroids. Exposure of the fetus to elevated levels of corticosteroids has been shown to play a role in programming of hypertension (25). Blood pressure responsiveness to ANG II, but not to noradrenaline, is enhanced in fetal sheep after cortisol infusion (43). In adult rats, glucocorticoids can increase pressor response to ANG II and AT1R expression on vascular smooth muscle cells (39). Whether vascular AT1R expression is increased from early in development is unknown.
Increased response to ANG II in the absence of endothelium raises the possibility that ANG II stimulates liberation of endothelial relaxing factor(s). Activation of AT1R located on endothelial cells can modulate the release of other vasoactive molecules such as nitric oxide (5), prostacyclin (11), and vasoconstrictor endothelin. We and others reported decreased vasorelaxation to nitric oxide-dependent mechanisms in programmed hypertension in vivo (34) and ex vivo (6, 16). Vasomotor response to prostacyclin was studied in adult LP and CTRL offspring pial microvessels and was found unaltered (16). In the current studies, vasoconstriction in response to ANG II was increased for the two diet groups in the endothelium-depleted compared with intact carotids. This suggests either that ANG II stimulates the release of vasorelaxant factors and/or that there is constitutive release of endothelial derived relaxing factors. If increased carotid vasomotor response to ANG II had been secondary to defective endothelium-derived relaxing factors, the difference between groups in their vasoconstriction to ANG II would have been less in the absence of endothelium. On the contrary, the increase in the vasoconstriction response was significantly more in LP offspring, suggesting that if some defective endothelium-mediated relaxation prevails, exaggerated response of vascular smooth muscle cells to ANG II predominates in LP carotid arteries rings. Carotid arteries are conductance and not resistance vessels, and properties of the different vascular beds or vessel type differ (29). Therefore the observation that defective endothelium-mediated vasorelaxation does not play a (major) role in the carotid responses to ANG II cannot be considered as contradictory with previously published studies (6, 16, 34).
ANG II can lead to the synthesis of endothelin (36). Studies realized in the presence of phosphoramidon suggest that endothelin does not play a role in ANG II-mediated vasoconstriction of the carotid arteries. These data are also in agreement with the observation that vasoconstriction was enhanced in the absence of endothelium: if endothelin had mediated part of the response observed to ANG II, vasoconstriction to ANG II would have decreased in the absence of endothelium.
Phosphoramidon also blocks neutral endopeptidase 24.11, which metabolizes ANG I to form ANG-(1–7). ANG-(1–7) can also be formed from ANG II under the action of ACE2. ANG-(1–7) through activation of the G protein-coupled Mas receptor can counteract the effects of AT1R and result in vasodilatation (9, 38). We observed no vasomotor effect of ANG-(1–7) in both groups, indicating that vasoconstriction in response to ANG II is not modulated by ANG-(1–7) and that Mas receptor seems absent from carotid arteries (38). We are not aware of studies demonstrating the presence (or absence) of Mas receptor in rat carotid arteries.
Animals and humans studies have shown that increased vascular reactive oxygen species (ROS), especially superoxide anion, contributes significantly to the vascular dysfunction present in hypertension (28). SOD mimetic can normalize blood pressure and regional blood flow in ANG II-infused rats (31). Normalization by SOD mimetic Tempol of the enhanced vasoconstriction to ANG II in LP offspring suggests indeed an increased vascular production of superoxide anion. This is supported by the lucigenin-enhanced chemiluminescence revealing a marked increase in the aortic production of superoxide anion in the presence of ANG II in the LP group only, which is inhibited by the addition of DPI. The latter element suggests that superoxide anion is generated through a flavin-containing enzyme. Among all the potential flavin-containing enzymatic sources of ROS, a functional role in adult hypertension has been recognized for NADH/NADPH oxidases, uncoupled nitric oxide synthase, and xanthine oxidase, as well as for the mitochondrial electron transport chain (7, 49). The nearly complete inhibition of superoxide production in the presence of apocynin indicates that NADPH oxidase is the main source of superoxide in LP offspring aorta, both in baseline conditions and after stimulation by ANG II. These results are in agreement with many reports showing that, in adults with chronic hypertension, membrane-bound NADH and NADPH oxidases are the most significant ROS source in the vascular wall (14, 49) and that ANG II enhances the vascular production of ROS (mostly superoxide) essentially through the activation of NADH and NADPH oxidases (44). In another model of fetal programming of hypertension, ANG II was shown to increase superoxide production through NAPDH oxidase in mesenteric arteries; the latter studies, however, did not examine the vasomotor response to ANG II (10), but it did show that apocynin normalized defective vasodilatation to bradykinin and ACh.
These results provide a basic mechanism by which increased vascular reactivity to ANG II prevails in adult LP offspring. However, it is unknown from our and previously published data whether oxidative stress was present early in life and could have initiated vascular dysfunction and hypertension. This can be hypothesized, considering the key role of ANG II in the development of “programmed” hypertension (see introduction). ANG II can increase NADPH oxidase activity and expression of its components (8, 12, 15, 28, 35). In turn, NADH and NADPH oxidase enhance the production of ROS through activation of xanthine oxidase, the auto-oxidation of NADH, and the inactivation of SOD (8, 12, 15, 28, 35). Hence, increased RAS activity contributes significantly to augmented ROS and could lead to vascular dysfunction and vascular structural changes such as microvascular rarefaction (34).
This work was supported by grants from the Canadian Institutes of Health Research (to F. Gobeil, Jr. and A. M. Nuyt), the Hospital for Sick Children Foundation (to P. Hardy and A. M. Nuyt) and the Heart and Stroke Foundation of Canada (to A. M. Nuyt). Patrick Pladys was supported by an Institut National de Santé et Recherche Médicale (INSERM, France) and the Canadian Institutes of Health Research CIHR research fellowship and by the Conseil Régional de Bretagne. Anne Monique Nuyt, Pierre Hardy, and Fernand Gobeil Jr. are recipients of Junior scholarship from the Fonds de la Recherche en Santé du Québec and researchers of the Canada Foundation for Innovation.
We thank Dr. Rhian M. Touyz for critical reading of the manuscript and helpful suggestions.
Current address for P. Pladys: Unité de médecine néonatale, Hôpital Sud, CHU Rennes, 16 Bd de Bulgarie, BP 90347, 35203 Rennes Cedex 2, France. Address of G. Cambonie: Service de réanimation pédiatrique et néonatale, Hôpital Arnaud de Villeneuve, Montpellier, France.
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