Experiments investigated maturation of endothelial function in the postnatal period. Carotid arteries isolated from newborn (postnatal day 1, P1) to P21 mice were assessed in myographs at transmural pressure (PTM) of 20 mmHg (P1 blood pressure, BP). Acetylcholine was ineffective in P1 but powerfully dilated P7 arteries, whereas NO-donor DEA-NONOate caused similar dilation at P1 and P7. Dilation to acetylcholine at P7 was abolished by inhibition of NO synthase (NOS) (l-NAME) or of phosphoinositide-3-kinase (PI3K) (wortmannin, LY294002). Endothelial NOS (eNOS) expression decreased in P7 compared with P1 arteries, although acetylcholine increased PO4-eNOS-Ser(1177) in P7 but not in P1 arteries. Endothelial maturation may therefore reflect increased signaling through PI3K, Akt, and eNOS. Systemic BP increases dramatically in the early postnatal period. After exposing P1 arteries to transient increased PTM (50 mmHg, 60 min), acetylcholine caused powerful dilation and increased PO4-eNOS-Ser(1177). Pressure-induced rescue of acetylcholine dilation was abolished by PI3K or NOS inhibition. Transient increased PTM did not affect dilation at P7, or dilation to NO-donor in P1 arteries. Width of endothelial adherens junctions (VE-cadherin immunofluorescence) increased significantly from P1 to P7, and in P1 arteries exposed to transient increased PTM. A function-blocking antibody to VE-cadherin reduced the pressure-induced rescue of acetylcholine responses at P1, and the dilation to acetylcholine in P7 arteries. Therefore, maturation of newborn endothelium dilator function may be induced by increasing BP in the postnatal period. Furthermore, this may be mediated by VE-cadherin signaling at adherens junctions. Interruption of this maturation pathway may contribute to developmental and adult vascular diseases.

Sexual dimorphism depends on sex-biased gene expression, but the contributions of microRNAs (miRNAs) have not been globally assessed. We therefore produced an extensive small RNA sequencing data set to analyze male and female miRNA expression profiles in mouse, opossum, and chicken. Our analyses uncovered numerous cases of somatic sex-biased miRNA expression, with the largest proportion found in the mouse heart and liver. Sex-biased expression is explained by miRNA-specific regulation, including sex-biased chromatin accessibility at promoters, rather than piggybacking of intronic miRNAs on sex-biased protein-coding genes. In mouse, but not opossum and chicken, sex bias is coordinated across tissues such that autosomal testis-biased miRNAs tend to be somatically male-biased, whereas autosomal ovary-biased miRNAs are female-biased, possibly due to broad hormonal control. In chicken, which has a Z/W sex chromosome system, expression output of genes on the Z Chromosome is expected to be male-biased, since there is no global dosage compensation mechanism that restores expression in ZW females after almost all genes on the W Chromosome decayed. Nevertheless, we found that the dominant liver miRNA, miR-122-5p, is Z-linked but expressed in an unbiased manner, due to the unusual retention of a W-linked copy. Another Z-linked miRNA, the male-biased miR-2954-3p, shows conserved preference for dosage-sensitive genes on the Z Chromosome, based on computational and experimental data from chicken and zebra finch, and acts to equalize male-to-female expression ratios of its targets. Unexpectedly, our findings thus establish miRNA regulation as a novel gene-specific dosage compensation mechanism.

Lindgren I, Altimiras J. Chronic prenatal hypoxia sensitizes ␤-adrenoceptors in the embryonic heart but causes postnatal desensitization.

We have previously demonstrated a programming effect of prenatal hypoxia on the cardiac ␤-adrenergic (␤AR) response. The aim of this study was to determine 1) whether the decrease in ␤AR sensitivity in prenatally hypoxic 5-wk old chicken hearts is linked to changes in ␤ 1AR/␤2ARs, G␣i expression and cAMP accumulation and 2) whether prenatal hypoxia has an effect on heart function in vivo. We incubated eggs in normoxia (N, 21% O 2) or hypoxia from day 0 (H, 14% O 2) and raised the posthatchlings to 5 wk of age. Cardiac ␤ 1AR/␤2ARs were assessed through competitive binding of [ 3 H]CGP-12177 with specific ␤1AR or ␤2AR blockers. G␣s and G␣i proteins were assessed by Western blot and cAMP accumulation by ELISA. Echocardiograms were recorded in anesthetized birds to evaluate diastolic/systolic diameter and heart rate and tissue sections were stained for collagen. We found an increase in relative heart mass, ␤ 1ARs, and G␣s in prenatally hypoxic hearts. cAMP levels after isoproterenol stimulation and collagen content was not changed in H compared with N, but in vivo echocardiograms showed systolic contractile dysfunction. The changes in ␤AR and G protein subtypes may be indicative of an early compensatory stage in the progression of cardiac dysfunction, further supported by the cardiac hypertrophy and systolic contractile dysfunction. We suggest that it is not the changes in the proximal part of the ␤AR system that causes the decreased cardiac contractility, but Ca 2ϩ handling mechanisms further downstream in the ␤AR signaling cascade.␤-adrenergic receptors; G proteins; developmental programming; systolic dysfunction; heart failure THE CONNECTION between low birth weight and increased risk for adult disease is well recognized, and many studies have confirmed the phenomenon of developmental (or fetal) programming of adult cardiovascular disease (1-3, 36). Cardiovascular disease is closely related to changes in ␤-adrenoceptor (␤AR) signaling in the heart (26), but not much is known about how the ␤AR system is affected by prenatal stress such as hypoxia. We previously showed that the response to vascular ␤AR is enhanced in embryos incubated in chronic prenatal hypoxia and that chronic prenatal hypoxia decreases ␤AR sensitivity to epinephrine stimulation in the 5-wk postnatal chicken heart, without changes in receptor density (18). This in vitro evidence indicates that prenatal hypoxia has a programming effect on ␤AR signaling in the adult, but the molecular background of the decreased ␤AR sensitivity in the heart and the in vivo implications of this loss of function are not known.Classic ␤AR signaling starts with a conformational change of the receptor in response to catecholamine stimulation. This enables intracellular association of stimulatory G proteins to the receptor that upon the interaction dissociates into an ␣-subunit (G ␣s ) and a ␤␥ complex. The G ␣s subunit in turn stimulates adenylyl cyclase (AC) to convert ATP to cAMP and cAMP act on kinases responsible for both altering calcium flux in the cell (leading t...

Lindgren I, Crossley II D, Villamor E, Altimiras J. Hypotension in the chronically hypoxic chicken embryo is related to the ␤-adrenergic response of chorioallantoic and femoral arteries and not to bradycardia. Am J Physiol Regul Integr Comp Physiol 301: R1161-R1168, 2011. First published July 27, 2011; doi:10.1152/ajpregu.00458.2010.-Prolonged fetal hypoxia leads to growth restriction and can cause detrimental prenatal and postnatal alterations. The embryonic chicken is a valuable model to study the effects of prenatal hypoxia, but little is known about its long-term effects on cardiovascular regulation. We hypothesized that chicken embryos incubated under chronic hypoxia would be hypotensive due to bradycardia and ␤AR-mediated relaxation of the systemic and/or the chorioallantoic (CA) arteries. We investigated heart rate, blood pressure, and plasma catecholamine levels in 19-day chicken embryos (total incubation 21 days) incubated from day 0 in normoxia or hypoxia (14 -15% O2). Additionally, we studied ␣-adrenoceptor (␣AR)-mediated contraction, relaxation to the ␤-adrenoceptor (␤AR) agonist isoproterenol, and relaxation to the adenylate cyclase activator forskolin in systemic (femoral) and CA arteries (by wire myography). Arterial pressure showed a trend toward hypotension in embryos incubated under chronic hypoxic conditions compared with the controls (mean arterial pressure 3.19 Ϯ 0.18 vs. 2.59 Ϯ 0.13 kPa, normoxia vs. hypoxia, respectively. P ϭ 0.056), without an accompanied bradycardia and elevation in plasma norepinephrine and lactate levels. All vessels relaxed in response to ␤AR stimulation with isoproterenol, but the CA arteries completely lacked an ␣AR response. Furthermore, hypoxia increased the sensitivity of femoral arteries (but not CA arteries) to isoproterenol. Hypoxia also increased the responsiveness of femoral arteries to forskolin. In conclusion, we suggest that hypotension in chronic hypoxic chicken embryos is the consequence of elevated levels of circulating catecholamines acting in vascular beds with exclusive (CA arteries) or exacerbated (femoral arteries) ␤AR-mediated relaxation, and not a consequence of bradycardia. prenatal hypoxia; hypoxic hypotension; chorioallantoic membrane; ␤-adrenoceptors; ␣-adrenoceptors