Long-term hypoxia uncouples Ca2+ and eNOS in bradykinin-mediated pulmonary arterial relaxation.

Bradykinin-induced activation of the pulmonary endothelium triggers a rise in intracellular Ca2+ that activates nitric oxide (NO)-dependent vasorelaxation. Chronic hypoxia is commonly associated with increased pulmonary vascular tone, which can cause pulmonary hypertension in responsive individuals. In the present study, we tested the hypothesis that long-term high-altitude hypoxia (LTH) diminishes bradykinin-induced Ca2+ signals and inhibits endothelial nitric oxide synthase (eNOS), prostacyclin (PGI2), and large-conductance K+ (BKCa) channels in sheep, which are moderately responsive to LTH, resulting in decreased pulmonary arterial vasorelaxation. Pulmonary arteries were isolated from ewes kept near sea level (720 m) or at high altitude (3,801 m) for >100 days. Vessel force was measured with wire myography and endothelial intracellular Ca2+ with confocal microscopy. eNOS was inhibited with 100 µM NG-nitro-l-arginine methyl ester (l-NAME), PGI2 production was inhibited with 10 µM indomethacin that inhibits cyclooxygenase, and BKCa channels were blocked with 1 mM tetraethylammonium. Bradykinin-induced endothelial Ca2+ signals increased following LTH, but bradykinin relaxation decreased. Furthermore, some vessels contracted in response to bradykinin after LTH. l-NAME sensitivity decreased, suggesting that eNOS dysfunction played a role in uncoupling Ca2+ signals and bradykinin relaxation. The Ca2+ ionophore A-23187 (10 µM) elicited an enhanced Ca2+ response following LTH while relaxation was unchanged although l-NAME sensitivity increased. Additionally, BKCa function decreased during bradykinin relaxation following LTH. Western analysis showed that BKCa α-subunit expression was increased by LTH while that for the ß1 subunit was unchanged. Overall, these results suggest that those even moderately responsive to LTH can have impaired endothelial function.

Long-term high-altitude hypoxia influences pulmonary arterial L-type calcium channel-mediated Ca2+ signals and contraction in fetal and adult sheep.

Long-term hypoxia (LTH) has a profound effect on pulmonary arterial vasoconstriction in the fetus and adult. Dysregulation in Ca2+ signaling is important during the development of LTH-induced pulmonary hypertension. In the present study, we tested the hypothesis that L-type Ca2+ channels (CaL), which are voltage dependent and found in smooth, skeletal, and cardiac muscle, are important in the adaptation of pulmonary arterial contractions in postnatal maturation and in response to LTH. Pulmonary arteries were isolated from fetal or adult sheep maintained at low or high altitude (3,801 m) for >100 days. The effects were measured using an L-type Ca2+ channel opener FPL 64176 (FPL) in the presence or absence of an inhibitor, Nifedipine (NIF) on arterial contractions, intracellular Ca2+ oscillations, and ryanodine receptor-driven Ca2+ sparks. FPL induced pulmonary arterial contractions in all groups were sensitive to NIF. However, when compared with 125 mM K+, FPL contractions were greater in fetuses than in adults. FPL reduced Ca2+ oscillations in myocytes of adult but not fetal arteries, independently of altitude. The FPL effects on Ca2+ oscillations were reversed by NIF in myocytes of hypoxic but not normoxic adults. FPL failed to enhance Ca2+ spark frequency and had little impact on spatiotemporal firing characteristics. These data suggest that CaL-dependent contractions are largely uncoupled from intracellular Ca2+ oscillations and the development of Ca2+ sparks. This raises questions regarding the coupling of pulmonary arterial contractility to membrane depolarization, attendant CaL facilitation, and the related associations with the activation of Ca2+ oscillations and Ca2+ sparks.

Sir Joseph Barcroft: one victorian physiologist's contributions to a half century of discovery.

During the first half of the 20th Century, Joseph Barcroft, KBE, FRS of Cambridge University became a world leader in respiratory physiology. He determined the role of neural stimulation in the oxygen consumption of several organs, established many of the factors that regulate the binding of oxygen to haemoglobin, explored the determinants of a human's acclimatization to high altitude and developed the field of fetal cardiovascular physiology. Chair of the Cambridge Department of Physiology from 1925 to 1937, he served as a consultant and member of many UK governmental committees. During World War I, he led a British research unit exploring the effects of poisonous gases on pulmonary function and related problems. In addition to his almost 300 publications, several of his monographs are considered as classics.

Developmental acceleration of bradykinin-dependent relaxation by prenatal chronic hypoxia impedes normal development after birth.

Bradykinin-induced activation of the pulmonary endothelium triggers nitric oxide production and other signals that cause vasorelaxation, including stimulation of large-conductance Ca(2+)-activated K(+) (BKCa) channels in myocytes that hyperpolarize the plasma membrane and decrease intracellular Ca(2+). Intrauterine chronic hypoxia (CH) may reduce vasorelaxation in the fetal-to-newborn transition and contribute to pulmonary hypertension of the newborn. Thus we examined the effects of maturation and CH on the role of BKCa channels during bradykinin-induced vasorelaxation by examining endothelial Ca(2+) signals, wire myography, and Western immunoblots on pulmonary arteries isolated from near-term fetal (∼ 140 days gestation) and newborn, 10- to 20-day-old, sheep that lived in normoxia at 700 m or in CH at high altitude (3,801 m) for >100 days. CH enhanced bradykinin-induced relaxation of fetal vessels but decreased relaxation in newborns. Endothelial Ca(2+) responses decreased with maturation but increased with CH. Bradykinin-dependent relaxation was sensitive to 100 µM nitro-L-arginine methyl ester or 10 µM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, supporting roles for endothelial nitric oxide synthase and soluble guanylate cyclase activation. Indomethacin blocked relaxation in CH vessels, suggesting upregulation of PLA2 pathways. BKCa channel inhibition with 1 mM tetraethylammonium reduced bradykinin-induced vasorelaxation in the normoxic newborn and fetal CH vessels. Maturation reduced whole cell BKCa channel α1-subunit expression but increased ß1-subunit expression. These results suggest that CH amplifies the contribution of BKCa channels to bradykinin-induced vasorelaxation in fetal sheep but stunts further development of this vasodilatory pathway in newborns. This involves complex changes in multiple components of the bradykinin-signaling axes.

Vitamin D status and metabolism in an ovine pregnancy model: effect of long-term, high-altitude hypoxia.

Vitamin D status increases during healthy mammalian pregnancy, but the molecular determinants remain uncharacterized. The first objective of this study was to determine the effects of pregnancy, and the second objective was to examine the role of chronic hypoxia on vitamin D status and metabolism in an ovine model. We analyzed the plasma levels of cholecalciferol, 25-OH-D, and 1α,25-(OH)2D in nonpregnant ewes, near-term pregnant ewes, and their fetuses exposed to normoxia (low altitude) or hypoxia (high-altitude) for 100 days. Hypoxic sheep had increased circulating levels of 25-OH-D and 1α,25-(OH)2D compared with normoxic sheep. Hypoxia increases in 25-OH-D were associated with increased expression of renal 25-hydroxylases CYP2R1 and CYP2J. Pregnancy did not increase further the plasma levels of 25-OH-D, but it significantly increased those of the active metabolite, 1α,25-(OH)2D, in both normoxic and hypoxic ewes. Increased bioactivation of vitamin D correlated with increased expression of the vitamin D-activating enzyme CYP27b1 and decreased expression of the inactivating enzyme CYP24a1 in maternal kidneys and placentas. Hypoxia increased parathyroid hormone levels and further increased renal CYP27b1. Pregnancy and hypoxia decreased the expression of vitamin D receptor (VDR) in maternal kidney and lung, with opposite effects on placental VDR. We conclude that ovine pregnancy is a model of increased vitamin D status, and long-term hypoxia further improves vitamin D status due to pregnancy- and hypoxia-specific regulation of VDR and metabolic enzymes.

Postprandial lipids accelerate and redirect nitric oxide consumption in plasma.

Nitric oxide (NO) and O2 are both three-to four-fold more soluble in biological lipids than in aqueous solutions. Their higher concentration within plasma lipids accelerates NO autoxidation to an extent that may be of importance to overall NO bioactivity. This study was undertaken to test the hypothesis that increased plasma lipids after a high-fat meal appreciably accelerate NO metabolism and alter the byproducts formed. We found that plasma collected from subjects after consumption of a single high-fat meal had a higher capacity for NO consumption and consumed NO more rapidly compared to fasting plasma. This increased NO consumption showed a direct correlation with plasma triglyceride concentrations (p = 0.006). The accelerated NO consumption in postprandial plasma was reversed by removal of the lipids from the plasma, was mimicked by the addition of hydrophobic micelles to aqueous buffer, and could not be explained by the presence of either free hemoglobin or ceruloplasmin. The products of NO consumption were shifted in postprandial plasma, with 55% more nitrite (n = 12, p = 0.002) but 50% less SNO (n = 12, p = 0.03) production compared to matched fasted plasma. Modeling calculations indicated that NO autoxidation was accelerated by about 48-fold in the presence of plasma lipids. We conclude that postprandial triglyceride-rich lipoproteins exert a significant influence on NO metabolism in plasma.

S-nitrosothiols dilate the mesenteric artery more potently than the femoral artery by a cGMP and L-type calcium channel-dependent mechanism.

S-nitrosothiols (SNOs) are metabolites of NO with potent vasodilatory activity. Our previous studies in sheep indicated that intra-arterially infused SNOs dilate the mesenteric vasculature more than the femoral vasculature. We hypothesized that the mesenteric artery is more responsive to SNO-mediated vasodilation, and investigated various steps along the NO/cGMP pathway to determine the mechanism for this difference. In anesthetized adult sheep, we monitored the conductance of mesenteric and femoral arteries during infusion of S-nitroso-l-cysteine (L-cysNO), and found mesenteric vascular conductance increased (137 ± 3%) significantly more than femoral conductance (26 ± 25%). Similar results were found in wire myography studies of isolated sheep mesenteric and femoral arteries. Vasodilation by SNOs was attenuated in both vessel types by the presence of ODQ (sGC inhibitor), and both YC-1 (sGC agonist) and 8-Br-cGMP (cGMP analog) mediated more potent relaxation in mesenteric arteries than femoral arteries. The vasodilatory difference between mesenteric and femoral arteries was eliminated by antagonists of either protein kinase G or L-type Ca(2+) channels. Western immunoblots showed a larger L-type Ca(2+)/sGC abundance ratio in mesenteric arteries than in femoral arteries. Fetal sheep mesenteric arteries were more responsive to SNOs than adult mesenteric arteries, and had a greater L-Ca(2+)/sGC ratio (p = 0.047 and r = -0.906 for correlation between Emax and L-Ca(2+)/sGC). These results suggest that mesenteric arteries, especially those in fetus, are more responsive to SNO-mediated vasodilation than femoral arteries due to a greater role of the L-type calcium channel in the NO/cGMP pathway.

Microarray gene expression analysis in ovine ductus arteriosus during fetal development and birth transition.

BACKGROUND: Patent ductus arteriosus (PDA) in the newborn is the most common congenital heart anomaly and is significantly more common in preterm infants. Contemporary pharmacological treatment is effective in only 70-80% of the cases. Moreover, indomethacin or ibuprofen, which are used to close a PDA may be accompanied by serious side effects in premature infants. To explore the novel molecular pathways, which may be involved in the maturation and closure of the ductus arteriosus (DA), we used fetal and neonatal sheep to test the hypothesis that maturational development of DA is associated with significant alterations in specific mRNA expression. METHODS: We conducted oligonucleotide microarray experiments on the isolated mRNA from DA and ascending aorta from three study groups (premature fetus-97 ± 0 d, near-term fetus-136 ± 0.8 d, and newborn lamb-12 ± 0 h). We compared the alterations in mRNA expression in DA and aorta to identify genes specifically involved in DA maturation. RESULTS: Results demonstrate significant changes in wingless-integrin1, thrombospondin 1, receptor activator of nuclear factor-kappa B, nitric oxide synthase, and retinoic acid receptor activation signaling pathways. CONCLUSION: We conclude that these pathways may play an important role during both development and postnatal DA closure and warrant further investigation.

Long-term hypoxia increases calcium affinity of BK channels in ovine fetal and adult cerebral artery smooth muscle.

Acclimatization to high-altitude, long-term hypoxia (LTH) reportedly alters cerebral artery contraction-relaxation responses associated with changes in K(+) channel activity. We hypothesized that to maintain oxygenation during LTH, basilar arteries (BA) in the ovine adult and near-term fetus would show increased large-conductance Ca(2+) activated potassium (BK) channel activity. We measured BK channel activity, expression, and cell surface distribution by use of patch-clamp electrophysiology, flow cytometry, and confocal microscopy, respectively, in myocytes from normoxic control and LTH adult and near-term fetus BA. Electrophysiological data showed that BK channels in LTH myocytes exhibited 1) lowered Ca(2+) set points, 2) left-shifted activation voltages, and 3) longer dwell times. BK channels in LTH myocytes also appeared to be more dephosphorylated. These differences collectively make LTH BK channels more sensitive to activation. Studies using flow cytometry showed that the LTH fetus exhibited increased BK ß1 subunit surface expression. In addition, in both fetal groups confocal microscopy revealed increased BK channel clustering and colocalization to myocyte lipid rafts. We conclude that increased BK channel activity in LTH BA occurred in association with increased channel affinity for Ca(2+) and left-shifted voltage activation. Increased cerebrovascular BK channel activity may be a mechanism by which LTH adult and near-term fetal sheep can acclimatize to long-term high altitude hypoxia. Our findings suggest that increasing BK channel activity in cerebral myocytes may be a therapeutic target to ameliorate the adverse effects of high altitude in adults or of intrauterine hypoxia in the fetus.

Antenatal maternal low protein diet: ACE-2 in the mouse lung and sexually dimorphic programming of hypertension.

Elevated blood pressure is an important global health problem, and in-utero under-nutrition may be an important factor in the pathogenesis of hypertension. In the present study, we tested the hypothesis that antenatal maternal low protein diet (MLPD) leads to sexually dimorphic developmental programming of the components of the pulmonary renin-angiotensin system. This may be important in the antenatal MLPD-associated development of hypertension. In pregnant mice, we administered normal (control) and isocaloric 50% protein restricted diet, commencing one week before mating and continuing until delivery of the pups. From the 18th to 24th week postnatal, we measured blood pressure in the offspring by use of a non-invasive tail-cuff method. In the same mice, we examined the mRNA and protein expression of the key components of the pulmonary renin-angiotensin system. Also, we examined microRNA complementary to angiotensin converting enzymes (ACE) 2 in the offspring lungs. Our results demonstrate that as a consequence of antenatal MLPD: 1) pup birthweight was significantly reduced in both sexes. 2) female offspring developed hypertension, but males did not. 3) In female offspring, ACE-2 protein expression was significantly reduced without any change in the mRNA levels. 4) miRNA 429, which has a binding site on ACE-2 - 3' UTR was significantly upregulated in the female antenatal MLPD offspring. 5) In males, ACE-2 mRNA and protein expression were unaltered. We conclude that in the mouse, antenatal MLPD-induced reduction of ACE-2 in the female offspring lung may be an important mechanisms in sexually dimorphic programming of hypertension.

Acclimatization to long-term hypoxia: gene expression in ovine carotid arteries.

Exposure to acute high-altitude hypoxia is associated with an increase in cerebral blood flow (CBF) as a consequence of low arterial O2 tension. However, in response to high altitude acclimatization, CBF returns to levels similar to those at sea level, and tissue blood flow is maintained by an increase in angiogenesis. Of consequence, dysregulation of the acclimatization responses and CBF can result in acute mountain sickness, acute cerebral and/or pulmonary edema. To elucidate the signal transduction pathways involved in successful acclimatization to high altitude, in ovine carotid arteries, we tested the hypothesis that high altitude-associated long-term hypoxia results in changes in gene expression of critical signaling pathways. We acclimatized nonpregnant adult sheep to 3,801 m altitude for ∼110 days and conducted oligonucleotide microarray experiments on carotid arteries. Of a total of 116 regulated genes, 58 genes were significantly upregulated and 58 genes were significantly downregulated (each >2-fold, P < 0.05). Major upregulated genes included suprabasin and myelin basic protein, whereas downregulated genes included BAG2. Several of these genes are known to activate the ERK canonical signal transduction pathway and the process of angiogenesis. We conclude that among other changes, the altered signal transduction molecules involved in high-altitude acclimatization are associated ERK activation and angiogenesis.

Characterization of an animal model of pregnancy-induced vitamin D deficiency due to metabolic gene dysregulation.

Vitamin D deficiency has been associated with pregnancy complications such as preeclampsia, gestational diabetes, and recurrent miscarriage. Therefore, we hypothesized differences in vitamin D status between healthy [Sprague-Dawley (SD) and Lewis (LW)] and complicated [Brown Norway (BN)] rat pregnancies. In SD, LW, and BN rats, we analyzed the maternal plasma levels of the vitamin D metabolites 25-OH-D and 1,25-(OH)2-D at prepregnancy, pregnancy, and postpartum. Analysis of the active metabolite 1,25-(OH)2-D showed a twofold increase in pregnant SD and LW rats but a nearly 10-fold decrease in pregnant BN rats compared with nonpregnant controls. BN rats had a pregnancy-dependent upregulation of CYP24a1 expression, a key enzyme that inactivates vitamin D metabolites. In contrast, the maternal renal expression of CYP24a1 in SD and LW rats remained constant throughout pregnancy. Analysis of the vitamin D receptor (VDR) indicated that LW and SD but not BN rats experience a pregnancy-induced 10-fold decrease in maternal renal VDR protein levels. Further analysis of bisulfite-converted and genomic DNA indicated that the observed differences in maternal renal regulation of CYP24a1 during pregnancy and lactation are not due to differences in CYP24a1 promoter methylation or single-nucleotide polymorphisms. Finally, supplementation with 1,25-(OH)2-D significantly improved the reproductive phenotype of BN rats by increasing litter size and maternal-fetal weight outcomes. We conclude that BN rats represent a novel animal model of pregnancy-specific vitamin D deficiency that is linked to pregnancy complications. Vitamin D deficiency in BN rats correlates with maternal renal CYP24a1 upregulation followed by CYP27b1 upregulation.

"Surprised by joy"*: four decades of contributions to developmental physiology.

Presented at the 40th Anniversary Celebration of the Center for Perinatal Biology of Loma Linda University School of Medicine, honoring Dr. Longo for his 40 years of extraordinary leadership and service, February 11, 2013.

Role of ceruloplasmin in nitric oxide metabolism in plasma of humans and sheep: a comparison of adults and fetuses.

Nitric oxide (NO) is metabolized in plasma, in part by the ferroxidase ceruloplasmin (Cp), to form nitrite and nitrosothiols (SNOs), which are proposed to mediate protective responses to hypoxia and ischemia. We hypothesized that NO metabolism would be attenuated in fetal plasma due to low Cp activity. We measured Cp concentrations and activity in plasma samples collected from adults and fetuses of humans and sheep. We then added NO ([NO]: 1.5 or 100 µM) to plasma and aqueous buffer and measured rates of NO disappearance and the production of nitrite and SNO. Cp concentrations in fetal plasma were <15% of adult levels. In aqueous buffer, 1.5 µM NO disappeared with a half-life of 347 ± 64 s (means ± SE) but in plasma of humans the half-life was 19 ± 2 s (adult) and 11 ± 1 s (fetus, P = 0.004) and in sheep it was 31 ± 3 s (adult) and 43 ± 5 s (fetus, P = 0.04). Cp activity was not correlated with the overall elimination half-life of NO or with the amount of SNO ([NO]: 100 µM) or nitrite ([NO]: 1.5 or 100 µM) produced but correlated with SNO yields at 1.5 µM [NO] (r = 0.92, P = 0.04). Our data demonstrate that Cp is not essential to the increased rate of metabolism of NO in plasma relative to aqueous buffers and that it is not essential to the production of nitrite from NO. Cp may be involved in the conversion of NO to SNO in plasma under near-physiological concentrations of NO.

Effect of chronic perinatal hypoxia on the role of rho-kinase in pulmonary artery contraction in newborn lambs.

Exposure to chronic hypoxia during gestation predisposes infants to neonatal pulmonary hypertension, but the underlying mechanisms remain unclear. Here, we test the hypothesis that moderate continuous hypoxia during gestation causes changes in the rho-kinase pathway that persist in the newborn period, altering vessel tone and responsiveness. Lambs kept at 3,801 m above sea level during gestation and the first 2 wk of life were compared with those with gestation at low altitude. In vitro studies of isolated pulmonary arterial rings found a more forceful contraction in response to KCl and 5-HT in high-altitude compared with low-altitude lambs. There was no difference between the effects of blockers of various pathways of extracellular Ca(2+) entry in low- and high-altitude arteries. In contrast, inhibition of rho-kinase resulted in significantly greater attenuation of 5-HT constriction in high-altitude compared with low-altitude arteries. High-altitude lambs had higher baseline pulmonary artery pressures and greater elevations in pulmonary artery pressure during 15 min of acute hypoxia compared with low-altitude lambs. Despite evidence for an increased role for rho-kinase in high-altitude arteries, in vivo studies found no significant difference between the effects of rho-kinase inhibition on hypoxic pulmonary vasoconstriction in intact high-altitude and low-altitude lambs. We conclude that chronic hypoxia in utero results in increased vasopressor response to both acute hypoxia and serotonin, but that rho-kinase is involved only in the increased response to serotonin.

Pulmonary artery smooth muscle cell proliferation and migration in fetal lambs acclimatized to high-altitude long-term hypoxia: role of histone acetylation.

High-altitude long-term hypoxia (LTH) is known to induce pulmonary arterial smooth muscle cell (PASMC) proliferation in the fetus, leading to pulmonary arterial remodeling and pulmonary hypertension of the newborn. The mechanisms underlying these conditions remain enigmatic however. We hypothesized that epigenetic alterations in fetal PASMC induced by high-altitude LTH may play an important role in modulating their proliferation during pulmonary arterial remodeling. To test this hypothesis, we have analyzed epigenetic alterations in the pulmonary vasculature of fetal lambs exposed to high-altitude LTH [pregnant ewes were kept at 3,801 m altitude from ~40 to 145 days gestation] or to sea level atmosphere. Intrapulmonary arteries were isolated, and fetal PASMC were cultured from both control and LTH fetuses. Compared with controls, in LTH fetus pulmonary arteries measurements of histone acetylation and global DNA methylation demonstrated reduced levels of global histone 4 acetylation and DNA methylation, accompanied by the loss of the cyclin-dependent kinase inhibitor p21. Treatment of LTH fetal PASMCs with histone deacetylase (HDAC) inhibitor trichostatin A decreased their proliferation rate, in part because of altered expression of p21 at both RNA and protein level. In PASMC of LTH fetuses, HDAC inhibition also decreased PDGF-induced cell migration and ERK1/2 activation and modulated global DNA methylation. On the basis of these observations, we propose that epigenetic alterations (reduced histone acetylation and DNA methylation) caused by chronic hypoxia leads to fetal PASMC proliferation and vessel remodeling associated with vascular proliferative disease and that this process is regulated by p21.

Maternal high-altitude hypoxia and suppression of ryanodine receptor-mediated Ca2+ sparks in fetal sheep pulmonary arterial myocytes.

Ca(2+) sparks are fundamental Ca(2+) signaling events arising from ryanodine receptor (RyR) activation, events that relate to contractile and dilatory events in the pulmonary vasculature. Recent studies demonstrate that long-term hypoxia (LTH) can affect pulmonary arterial reactivity in fetal, newborn, and adult animals. Because RyRs are important to pulmonary vascular reactivity and reactivity changes with ontogeny and LTH we tested the hypothesis that RyR-generated Ca(2+) signals are more active before birth and that LTH suppresses these responses. We examined these hypotheses by performing confocal imaging of myocytes in living arteries and by performing wire myography studies. Pulmonary arteries (PA) were isolated from fetal, newborn, or adult sheep that lived at low altitude or from those that were acclimatized to 3,801 m for > 100 days. Confocal imaging demonstrated preservation of the distance between the sarcoplasmic reticulum, nucleus, and plasma membrane in PA myocytes. Maturation increased global Ca(2+) waves and Ca(2+) spark activity, with sparks becoming larger, wider, and slower. LTH preferentially depressed Ca(2+) spark activity in immature pulmonary arterial myocytes, and these sparks were smaller, wider, and slower. LTH also suppressed caffeine-elicited contraction in fetal PA but augmented contraction in the newborn and adult. The influence of both ontogeny and LTH on RyR-dependent cell excitability shed new light on the therapeutic potential of these channels for the treatment of pulmonary vascular disease in newborns as well as adults.

Ovine middle cerebral artery characterization and quantification of ultrastructure and other features: changes with development.

Regulation of tone, blood pressure, and blood flow in the cerebral vasculature is of vital importance, particularly in the developing infant. We tested the hypothesis that, in addition to accretion of smooth muscle cells (SMCs) in cell layers with vessel thickening, significant changes in smooth muscle structure, as well as phenotype, extracellular matrix, and membrane proteins, in the media of cerebral arteries (CAs) during the course of late fetal development account for associated changes in contractility. Using transmission electron, confocal, wide-field epifluorescence, and light microscopy, we examined the structure and ultrastructure of CAs. Also, we utilized wire myography, Western immunoblotting, and real-time quantitative PCR to examine several other features of these arteries. We compared the main branch ovine middle CAs of 95- and 140-gestational day (GD) fetuses with those of adults (n = 5 for each experimental group). We observed a graded increase in phenylephrine- and KCl-induced contractile responses with development. Structurally, lumen diameter, media thickness, and media cross-sectional area increased dramatically from one age group to the next. With maturation, the cross-sectional profiles of CA SMCs changed from flattened bands in the 95-GD fetus to irregular ovoid-shaped fascicles in the 140-GD fetus and adult. We also observed a change in the type of collagen, specific integrin molecules, and several other parameters of SMC morphology with maturation. Ovine CAs at 95 GD appeared morphologically immature and poorly equipped to respond to major hemodynamic adjustments with maturation.

Gene expression in sheep carotid arteries: major changes with maturational development.

BACKGROUND: With development from immature fetus to near-term fetus, newborn, and adult, the cerebral vasculature undergoes a number of fundamental changes. Although the near-term fetus is prepared for a transition from an intra- to extra-uterine existence, this is not necessarily the case with the premature fetus, which is more susceptible to cerebrovascular dysregulation. In this study, we tested the hypothesis that the profound developmental and age-related differences in cerebral blood flow are associated with significant underlying changes in gene expression. METHODS: With the use of oligonucleotide microarray and pathway analysis, we elucidated significant changes in the transcriptome with development in sheep carotid arteries. RESULTS: As compared with adult, we demonstrate a U-shaped relationship of gene expression in major cerebrovascular network/pathways during early life, e.g., the level of gene expression in the premature fetus and newborn is considerably greater than that of the near-term fetus. Specifically, cell proliferation, growth, and assembly pathway genes were upregulated during early life. In turn, as compared with adult, mitogen-activated protein kinase-extracellular regulated kinase, actin cytoskeleton, and integrin-signaling pathways were downregulated during early life. CONCLUSION: In cranial vascular smooth muscle, highly significant changes occur in important cellular and signaling pathways with maturational development.

alpha(1)-Adrenergic receptor subtype function in fetal and adult cerebral arteries.

In the developing fetus, cerebral artery (CA) contractility demonstrates significant functional differences from that of the adult. This may be a consequence of differential activities of alpha(1)-adrenergic receptor (alpha(1)-AR) subtypes. Thus we tested the hypothesis that maturational differences in adrenergic-mediated CA contractility are, in part, a consequence of differential expression and/or activities of alpha(1)-AR subtypes. In CA from fetal ( approximately 140 days) and nonpregnant adult sheep, we used wire myography and imaging, with simultaneous measurement of tension and intracellular Ca(2+) concentration ([Ca(2+)](i)), radioimmunoassay, and Western immunoblots to examine phenylephrine (Phe)-induced contractile responses. The alpha(1A)-AR antagonists (5-MU and WB-4101) completely inhibited Phe-induced contraction in adult but not fetal CA; however, [Ca(2+)](i) increase was reduced significantly in both age groups. The alpha(1D)-AR antagonist (BMY-7378) blocked both Phe-induced contractions and Ca(2+) responses to a significantly greater extent in adult compared with fetal CA. In both age groups, inhibition of alpha(1A)-AR and alpha(1B)-AR, but not alpha(1D)-AR, significantly reduced inositol 1,4,5-trisphosphate responses to Phe. Western immunoblots demonstrated that the alpha(1)-AR subtype expression was only approximately 20% in fetal CA compared with the adult. Moreover, in fetal CA, the alpha(1D)-AR was expressed significantly greater than the other two subtypes. Also, in fetal but not adult CA, Phe induced a significant increase in activated ERK1/2; this increase in phosphorylated ERK was blocked by alpha(1B)-AR (CEC) and alpha(1D)-AR (BMY-7378) inhibitors, but not by alpha(1A)-AR inhibitors (5-MU or WB-4101). In conclusion, in the fetal CA, alpha(1B)-AR and alpha(1D)-AR subtypes play a key role in contractile response as well as in ERK activation. We speculate that in fetal CA alpha(1B)-AR and alpha(1D)-AR subtypes may be a critical factor associated with cerebrovascular growth and function.