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Biomedical subjects

I M Bird

Publications and source records attributed to I M Bird.

At least 19 recordsLinked to original sources
PubMed2001

Ovine caveolin-1: cDNA cloning, E. coli expression, and association with endothelial nitric oxide synthase.

Caveolin-1 (Cav-1), the principal coat protein of caveolae, plays an obligatory role in regulating the activity of endothelial nitric oxide (NO) synthase (eNOS). We propose that Cav-1 may be critical to eNOS-NO mediated uterine vasodilatation during pregnancy and estrogen replacement therapy. To test this hypothesis in the sheep model, we isolated the full-length cDNA of ovine Cav-1 (oCav-1) from a Lambda ZAP cDNA library of ovine placental artery endothelial cells. Thirty-two positive oCav-1 clones were recognized by a partial oCav-1 cDNA from this library, of which eight were sequenced. Restriction digestion of these clones revealed that the cDNAs of oCav-1 ranged from approximately 2.1 to 2.7 kb. Northern analysis of Cav-1 mRNAs in ovine uterine artery endothelial cells (UAEC) showed two transcripts of approximately 2.1 and 2.7 kb, respectively. Immunoreactive Cav-1 protein, but not caveolin-2 or caveolin-3, was detected in UAEC. Sequence analysis revealed that in addition to a 537-bp open reading frame encoding a 178 amino acid oCav-1 protein, full-length oCav-1 cDNAs apparently possess a approximately 1.6-2.1 kb 3'-untranslated region. Database searches with oCav-1 cDNA revealed that the coding region of mammalian Cav-1 genes is highly conserved. We prepared a recombinant full-length oCav-1 protein in which six consecutive histidine residues were tagged at the end of its COOH-terminus and developed a [His]6-tagged oCav-1 'pull-down assay' for studying the association of eNOS with Cav-1. Incubation of exogenous [His]6-tagged oCav-1 with resting UAEC extracts led to the formation of a [His]6-tagged oCav-1-eNOS complex. In the presence of a synthetic caveolin-scaffolding domain (CSD, aa 82-101) peptide, but not a mutated CSD peptide, [His]6-tagged oCav-1 associated eNOS was dose (0-10 microM)-dependently inhibited. eNOS association with Cav-1 in UAEC was further confirmed by the facts that eNOS co-immunoprecipitated with Cav-1 and vice versa, and that eNOS co-existed with Cav-1 during the isolation of caveolae membranes. Because dissociation of eNOS from Cav-1 is required for the activation of eNOS, eNOS association with Cav-1 in UAEC suggests an important role of Cav-1 in regulating UAEC production of NO and possibly NO-mediated uterine vasodilatation.

Animals↗
PubMed2001

Endothelium-derived nitric oxide synthase protein expression in ovine placental arteries.

During the third trimester, fetoplacental and uterine blood flows increase dramatically to meet the high metabolic demands of the growing fetus. We hypothesized that the expression of endothelial nitric oxide synthase (eNOS) in fetoplacental artery endothelium and the concentrations of nitric oxide (NO) and cyclic GMP (cGMP) in amniotic fluid (AF) are increased during the third trimester of ovine gestation. Placental arteries and AF were collected from ewes at 110, 120, 130, and 142 days of gestation (n = 24; mean +/- SEM term = 145 +/- 3 days). Expression of eNOS protein was measured in intact and denuded placental arteries and in endothelium-derived protein by Western analysis and confirmed by immunohistochemistry. Concentrations of NO (nitrates plus nitrites) and cGMP were determined in AF. Placental artery eNOS protein expression was localized to the endothelium, where it was markedly greater than in vascular smooth muscle. Placental artery endothelium-derived eNOS expression and AF cGMP concentrations were similar at 110 and 120 days of gestation; however, both peaked at 130 days at levels two- to threefold above baseline (P < 0.05) before returning to baseline at 142 days of pregnancy. The AF NO (nitrates plus nitrites) levels, however, increased progressively between 120 days of gestation and term (P < 0.05). We concluded that endothelium-derived placental artery eNOS levels, AF NO (nitrates plus nitrites), and AF cGMP were markedly increased during the third trimester, thus supporting a role for NO-mediated elevations in cGMP in the control of fetoplacental blood flow.

Amniotic Fluid↗
PubMed2001

Upregulation of eNOS in pregnant ovine uterine arteries by chronic hypoxia.

We tested the hypothesis that chronic high-altitude (3,820 m) hypoxia during pregnancy was associated with the upregulation of endothelial nitric oxide (NO) synthase (eNOS) protein and mRNA in ovine uterine artery endothelium and enhanced endothelium-dependent relaxation. In pregnant sheep, norepinephrine-induced dose-dependent contractions were increased by removal of the endothelium in both control and hypoxic uterine arteries. The increment was significantly higher in hypoxic tissues. The calcium ionophore A23187-induced relaxation of the uterine artery was significantly enhanced in hypoxic compared with control tissues. However, sodium nitroprusside- and 8-bromoguanosine 3',5'-cyclic monophosphate-induced relaxations were not changed. Accordingly, chronic hypoxia significantly increased basal and A23187-induced NO release. Chronic hypoxia increased eNOS protein and mRNA levels in the endothelium from uterine but not femoral or renal arteries. In nonpregnant animals, chronic hypoxia increased eNOS mRNA in uterine artery endothelium but had no effects on eNOS protein, NO release, or endothelium-dependent relaxation. Chronic hypoxia selectively augments pregnancy-associated upregulation of eNOS gene expression and endothelium-dependent relaxation of the uterine artery.

Altitude Sickness↗
PubMed2001

Endothelial vasodilator production by uterine and systemic arteries. VI. Ovarian and pregnancy effects on eNOS and NO(x).

Normal pregnancy and the follicular phase of the ovarian cycle are both estrogen-dominated physiological states that are characterized by elevations in uterine blood flow and endothelial nitric oxide synthase (eNOS) protein expression in the uterine artery (UA) endothelium. It is unknown if elevations in mRNA level account for the changes in protein or eNOS activity. We tested the hypothesis that pregnancy and the follicular phase are associated with increases in eNOS mRNA and the consequent elevated expression of eNOS protein results in increased circulating nitric oxide (NO) levels. UA were obtained from pregnant (PREG; n = 8; 110-130 days gestation; term = 145 +/- 3 days), nonpregnant luteal (LUT; n = 6), nonpregnant follicular (FOL; n = 6), and nonpregnant ovariectomized (OVEX; n = 6) sheep. Circulating NO levels were analyzed as total NO(2)-NO(3) (NO(x)). Western analysis performed on UA endothelial-isolated proteins demonstrated that eNOS protein levels were OVEX = LUT < or = FOL < PREG (P < 0.05), whereas eNOS mRNA expression (RT-PCR) in UA endothelial cells obtained by limited collagenase digestion was OVEX < LUT < FOL < PREG (P < 0.05). Pregnancy dramatically elevated eNOS protein (4.1- to 6.9-fold) and mRNA (2.4- to 6.9-fold) over LUT controls (P < 0.01). Circulating NO(x) levels were not altered by ovariectomy or the ovarian cycle but were elevated from 4.4 +/- 1.1 microM in LUT to 12 +/- 4, 22 +/- 3, and 41 +/- 3 microM at 110, 120, and 130 days gestation (P < 0.01). Systemic NO(x) levels in singleton (12.5 +/- 1.6 microM) were less (P < 0.01) than in multiple (twin 27.6 +/- 6.5 microM; triplet = 46 +/- 10 microM) pregnancies. Therefore, the follicular phase and, to a much greater extent, pregnancy are associated with elevations in UA endothelium-derived eNOS expression, although significant increases in systemic NO(x) levels were only observed in the PREG group (multiple > singleton). Thus, although UA endothelial increases in eNOS protein and mRNA levels are associated with high estrogen states, increases in local UA NO production may require additional eNOS protein activation to play its important role in the maintenance of uterine blood flow in pregnancy.

Animals↗
PubMed2001

Endothelial vasodilator production by uterine and systemic arteries. VII. Estrogen and progesterone effects on eNOS.

Uterine blood flow (UBF) and uterine artery endothelial nitric oxide synthase (eNOS) expression are greatest during the follicular vs. luteal phase. 17 beta-Estradiol (E(2)beta) increases UBF and elevates eNOS in ovine uterine but not systemic arteries; progesterone (P(4)) effects on E(2)beta changes of eNOS remain unclear. Nonpregnant ovariectomized sheep received either vehicle (n = 10), P(4) (0.9 g Controlled Internal Drug Release vaginal implants; n = 13), E(2)beta (5 microg/kg bolus + 6 microg x kg(-1) x day(-1); n = 10), or P(4) + E(2)beta (n = 12). Reproductive (uterine/mammary) and nonreproductive (omental/renal) artery endothelial proteins were procured on day 10, and eNOS was measured by Western analysis. P(4) and E(2)beta alone and in combination increased (P < 0.05) eNOS expression in uterine artery endothelium (vehicle = 100 +/- 16%, P(4) = 251 +/- 59%, E(2)beta = 566 +/- 147%, P(4) + E(2)beta = 772 +/- 211% of vehicle). Neither omental, renal, nor mammary artery eNOS was altered, demonstrating the local nature of steroid-induced maintenance of uterine arterial eNOS. In the myometrial microvasculature, eNOS was increased slightly (P = 0.06) with E(2)beta and significantly with P(4) + E(2)beta. Systemic NO(x) was increased with P(4) and P(4) + E(2)beta, but not E(2)beta, suggesting differential regulation of eNOS expression and activity, since P(4) increased eNOS in uterine artery endothelium while E(2)beta and the combination further increased eNOS protein.

Animals↗
PubMed2001

Zonal expression of endothelial nitric oxide synthase in sheep and rhesus adrenal cortex.

There is mounting evidence that nitric oxide (NO) may inhibit adrenal steroidogenesis by binding to the heme group of P450 enzymes, particularly the rate-limiting steps cholesterol side-change cleavage P450, aldosterone synthase P450, and 17 alpha-hydroxylase/C(17/20)-lyase P450. Using immunohistochemistry, nitrotyrosine was detectable throughout the ovine adrenal cortex, and endothelial NO synthase (eNOS) was further identified in zona glomerulosa (ZG) and at a higher level throughout the zona fasciculata, increasing toward the medulla. Caveolin-1, 90-kDa heat shock protein, ERK-1/2, and Akt, all known and proposed regulators of eNOS activity, were detected throughout the ovine adrenal cortex. Western immunoblotting confirmed the identity of these proteins as well as the absence of neuronal NOS, inducible NOS, caveolin-2, and caveolin-3. Through dual immunostaining we further identified for the first time a zona intermedia without strong staining for 17 alpha-hydroxylase/C(17/20)-lyase P450 or angiotensin II type 1 receptor, but positive for eNOS. Rhesus adrenals also stained positively for eNOS, but staining was seen only in the ZG and zona reticularis. We conclude that eNOS may play a role in controlling zone-specific aldosterone synthase vs. 11 beta-hydroxylase activities of the single CYP11B gene in sheep. In the rhesus monkey, NO may modulate ZG aldosterone synthase, but it is not needed for control of the distinct 11 beta-hydroxylase in the zona fasciculata. In the zona reticularis, however, eNOS may control C(19) steroid production at the level of 17 alpha-hydroxylase vs. 17,20-lyase activity otherwise unopposed by 3beta-hydroxysteroid dehydrogenase.

Adrenal Cortex↗
PubMed2001

Pregnancy-specific enhancement of agonist-stimulated ERK-1/2 signaling in uterine artery endothelial cells increases Ca(2+) sensitivity of endothelial nitric oxide synthase as well as cytosolic phospholipase A(2).

Uterine artery endothelial cells (UAEC) from pregnant ewes (P-UAEC) demonstrate generally enhanced ability to couple growth factor and G protein-coupled receptors to the ERK-1/2 signaling pathway and stimulate NO production independently of elevated [Ca(2+)]. Herein we investigate the signaling and vasodilator responses to ATP, an agonist that also elevates [Ca(2+)](i) in both NP and P-UAEC, to determine the relative importance of Ca(2+) vs. ERK-1/2 in the activation of eNOS. We observed in both NP-UAEC and P-UAEC that ATP acts through G protein-coupled P(2Y) receptors to activate phospholipase C and dose-dependently elevate [Ca(2+)](i) independently of extracellular Ca(2+). The small reduction in the [Ca(2+)](i) response in NP vs. P-UAEC did not, however, account for the difference in NO production by P-UAEC>>NP-UAEC. ATP had no stimulatory effect on Akt phosphorylation but rapidly stimulated ERK-1/2 phosphorylation in P-UAEC>>NP-UAEC in a manner that correlated with NO production. In both NP- and P-UAEC, both ERK-1/2 and Ca(2+) were absolutely required for eNOS as well as cPLA(2) activation and the Ca(2+) sensitivity of eNOS was enhanced through the cytosolic [Ca(2+)](i) range in P-UAEC>>NP-UAEC. Thus ERK-1/2 may regulate the Ca(2+) sensitivity of eNOS to an even greater extent than is known to occur for cPLA(2).

Animals↗
PubMed2000

Endothelial vasodilator production by uterine and systemic arteries. V. Effects of ovariectomy, the ovarian cycle, and pregnancy on prostacyclin synthase expression.

Prostacyclin (PGI(2)) is a potent vasodilator, the level of which is increased during pregnancy, and is the main eicosanoid of which production is elevated in the endothelium and vascular smooth muscle (VSM) of both uterine and omental (systemic) arteries. We tested the hypothesis that during physiologic states that have high uterine blood flow, such as pregnancy and the follicular phase of the ovarian cycle (versus luteal phase and ovariectomized ewes), there is an increased level of prostacyclin synthase (PGIS) expression in ovine uterine and omental artery endothelium and VSM. To investigate this, the cellular localization and PGIS protein expression level in uterine and systemic arteries was examined by immunohistochemistry as well as by Western immunoblot analysis of endothelial-isolated protein and denuded vessels (VSM). Whole uterine, but not omental (systemic), arteries from the pregnant ewes showed an increase (P < 0.001) in PGIS expression. Further localization of PGIS protein by immunohistochemistry and quantification by Western analysis showed PGIS to be somewhat higher in the uterine artery VSM (69 +/- 7%) than endothelium (31 +/- 7%). PGIS protein levels in uterine and omental artery endothelial isolated protein were not altered by ovariectomy or the ovarian cycle, although they were both significantly elevated by pregnancy. Uterine and omental artery VSM PGIS expression levels also were not altered by ovariectomy or the ovarian cycle, whereas PGIS expression, in uterine but not omental artery VSM showed a significant elevation during pregnancy. Thus, the rise in PGI(2) production by uterine arteries observed in ovine pregnancy is paralleled by an elevation in PGIS expression in both endothelium and VSM, whereas those seen in omental arteries is associated with increases in endothelial PGIS.

Animals↗
PubMed2000

Ontogeny of angiotensin II type 1 receptor and cytochrome P450(c11) in the sheep adrenal gland.

In the present study we investigated the ontogeny of the expression of the type 1 angiotensin receptor (AT(1)R mRNA) and the zonal localization of AT(1)R immunoreactivity (AT(1)R-ir) and cytochrome P450(c11) (CYP11B-ir) in the sheep adrenal gland. In the adult sheep and in the fetus from as early as 90 days gestation, intense AT(1)R-ir was observed predominantly in the zona glomerulosa and to a lesser extent in the zona fasciculata, and it was not detectable in the adrenal medulla. AT(1)R mRNA decreased 4-fold between 105 days and 120 days, whereas AT(1)R mRNA levels remained relatively constant between 120 days and the newborn period. In contrast, both in the adult sheep and in the fetal sheep from as early as 90 days gestation, intense CYP11B-ir was consistently detected throughout the adrenal cortex and in steroidogenic cells that surround the central adrenal vein. In conclusion, we speculate that the presence of AT(1)R in the zona fasciculata, and the higher levels of expression of AT(1)R at around 100 days gestation, may suggest that suppression of CYP17 is mediated via AT(1)R at this time. The abundant expression of AT(1)R-ir and CYP11B-ir in the zona glomerulosa of the fetal sheep adrenal gland would also suggest that lack of angiotensin II stimulation of aldosterone secretion is not due to an absence of AT(1)R or CYP11B in the zona glomerulosa.

Adrenal Glands↗
PubMed2000

Endothelial vasodilator production by uterine and systemic arteries. IV. Cyclooxygenase isoform expression during the ovarian cycle and pregnancy in sheep.

Uterine artery endothelial production of the potent vasodilator, prostacyclin, is greater in pregnant versus nonpregnant sheep and in whole uterine artery from intact versus ovariectomized ewes. We hypothesized that uterine artery cyclooxygenase (COX)-1 and/or COX-2 expression would be elevated during pregnancy (high estrogen and progesterone) and the follicular phase of the ovarian cycle (high estrogen/low progesterone) as compared to that in luteal phase (low estrogen/high progesterone) or in ovariectomized (low estrogen and progesterone) ewes. Uterine and systemic (omental) arteries were obtained from nonpregnant luteal-phase (LUT; n = 10), follicular-phase (FOL; n = 11), and ovariectomized (OVEX; n = 10) sheep, as well as from pregnant sheep (110-130 days gestation; term = 145 +/- 3 days; n = 12). Endothelial and vascular smooth muscle (VSM) COX-1 protein levels and uterine artery endothelial cell COX-1 mRNA levels were compared. Using immunohistochemistry and Western analysis, the primary location of COX-1 protein was the endothelium; that is, we observed 2.2-fold higher COX-1 protein levels in intact versus endothelium-denuded uterine artery and a 6.1-fold higher expression in the endothelium versus VSM (P < 0.05). COX-2 protein expression was not detectable in either uterine artery endothelium or VSM. COX-1 protein levels were observed to be higher (1.5-fold those of LUT) in uterine artery endothelium from FOL versus either OVEX or LUT nonpregnant ewes (P < 0.05), with substantially higher COX-1 levels seen in pregnancy (4.8-fold those of LUT). Increases in uterine artery endothelial COX-1 protein were highly correlated to increases in the level of COX-1 mRNA (r(2) = 0.66; P < 0.01) for all treatment groups (n = 6-8 per group), suggesting that increased COX-1 protein levels are regulated at the level of increased COX-1 mRNA. No change in COX-1 expression was observed between groups in a systemic (omental) artery. In conclusion, COX-1 expression is specifically up-regulated in the uterine artery endothelium during high uterine blood flow states such as the follicular phase and, in particular, pregnancy.

Animals↗
PubMed2000

Pregnancy-dependent changes in cell signaling underlie changes in differential control of vasodilator production in uterine artery endothelial cells.

During pregnancy, the uterine vasculature shows a marked increase in vasodilator production [prostacyclin (PGI2) and nitric oxide (NO)] in response to a number of agonists including angiotensin II (AII) and ATP. As a consequence vascular resistance is kept low, and uterine blood flow is maximized to meet the needs of the growing fetus. Studies of the molecular basis underlying this change in control of endothelial NO and PGI2 production have been hampered by the lack of availability of a suitable cell model. To that end we have developed and characterized a new ovine uterine artery endothelial cell (UAEC) culture model derived from nonpregnant (NP) or pregnant (P) ewes. Endothelial cells were isolated from pregnant (120-130 days; n = 6) and nonpregnant (n = 4) ewes and maintained in primary culture. Endothelial cells at passage 4 showed uniform expression of endothelial nitric oxide synthase (eNOS; an endothelial marker) as well as AII type 1 receptor and growth factor receptors and uniform uptake of acetylated low density lipoprotein (a property of endothelial cells not shared by fibroblasts or vascular smooth muscle cells), thus demonstrating cell purity. Expressions of eNOS, cyclooxygenase-1, PGI2 synthase, cytosolic phospholipase A2, AII type 1 receptor, and growth factor receptors are also maintained at passage 4. Mitogenesis is maintained in response to basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) in both NP-UAEC and P-UAEC. The differential production of vasodilators by NP-UAEC and P-UAEC is maintained in a manner similar to that previously reported in vivo. Thus, P-UAEC make NO in response to AII, ATP, bFGF, EGF, and VEGF, whereas NP-UAEC make NO in response to bFGF, EGF, and VEGF only. Similarly, P-UAEC make PGI2 in response to AII, ATP, bFGF, and VEGF, whereas NP-UAEC make PGI2 only in response to ATP and VEGF. As both cytosolic phospholipase A2 and eNOS may be regulated by both Ca2+ and protein kinases, we investigated the effects of these agonists on Ca2+ mobilization and ERK-1/2 phosphorylation. ATP consistently elevates Ca2+ levels in both P-UAEC and NP-UAEC. All other agonists were without acute (0-4 min) effect on Ca2+ in P-UAEC or NP-UAEC. In contrast, all agonists stimulated an acute (10 min) phosphorylation of ERK-1/2 in P-UAEC, whereas only EGF stimulated activation in NP-UAEC. P-UAEC production of PGI2 by agonists of both heptahelical receptors and growth factor receptors correlates closely with ERK-2 phosphorylation alone. For NO, this correlation holds for heptahelical receptor agonists, but additional signaling pathways are also implicated for bFGF and VEGF. In contrast, in NP-UAEC the lack of ERK-2 phosphorylation in response to all agonists other than EGF, and the dissociation between NO or PGI2 production and ERK-2 phosphorylation suggest that alternate pathways play a predominant role.

Angiotensin I↗
PubMed2000

Isolation of a full length ovine angiotensin II type-1 receptor (AT1-R) cDNA.

A full-length ovine AT1-R cDNA (2358 bases: Genebank AF254119) was isolated from ovine adrenal cortex using 3'-RACE. While homology of the 5'-untranslated region to human sequence was low (34.2%), the beginning of both human exon 1 and human exon 5 (encoding the end of the 5'-untranslated sequence/complete protein coding region/3'-untranslated sequence) were exceptionally conserved and in context. The intervening untranslated sequence showed lower homology to human sequence, but still contained four additional ATG sequences close to corresponding "in frame" TGA Stop codons shown in human to impair AT1-R translation in vitro. The putative protein coding sequence was >99% identical to the previous reported ovine genomic sequence. The predicted amino acid sequence in turn encoded a protein with the properties of a seven alpha-helix transmembrane receptor sharing closest homology (99.1%) to the bovine receptor and lowest to the rat Type la (90.5%). The 3'-untranslated region showed relatively high homology to the porcine and bovine receptor cDNA, but did not share the additional 643 bases found only in the bovine 3'-untranslated region. The ovine 3'-sequence included a polyadenylation signal as well as three AUUUA destabilization sequences observed in most other species including human. Thus ovine AT1-R mRNA stability may be short lived, and control of degradation may be an additional mechanism for regulation of ovine AT1-R expression.

3' Untranslated Regions↗
PubMed1999

Repeated use of betamethasone in rabbits: effects of treatment variation on adrenal suppression, pulmonary maturation, and pregnancy outcome.

OBJECTIVE: The aim of the study was to determine whether reduced birth weight, adrenal suppression, and lung maturation occur in parallel and are cumulative with increasing courses of betamethasone. STUDY DESIGN: Time-bred rabbits were assigned to a control group or to receive saline solution or 1, 2, or 3 courses of betamethasone (early treatment, beginning day 19). Two additional groups (n = 5 per group) were given 1 or 2 late courses (late treatment). Birth weight, serum cortisol, adrenal 17alpha-hydroxylase (P450c17) messenger ribonucleic acid and fetal lung surfactant proteins A and B were quantified on day 27. RESULTS: Fetal weight was inversely proportional to the number of courses, with late treatment having a greater effect. Maternal cortisol and P450c17 levels were progressively suppressed with each early course, but fetal cortisol and P450c17 levels were only suppressed after 3 courses. A single late treatment profoundly suppressed both maternal and fetal cortisol and P450c17 messenger ribonucleic acid levels. In contrast, fetal lung surfactant proteins A and B increased progressively with betamethasone courses, regardless of timing. CONCLUSIONS: Time from last injection to delivery determined adrenal suppression, whereas total betamethasone courses determined surfactant protein production. Lower birth weight was dependent on the number of courses and was greater with late treatment.

3-Hydroxysteroid Dehydrogenases↗
PubMed1999

Effects of angiogenic growth factors on endothelium-derived prostacyclin production by ovine uterine and placental arteries.

Uteroplacental and fetoplacental arteries produce substantial amounts of prostacyclin (PGI2). Because angiogenic growth factors such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF) are increased in pregnancy, we hypothesized that treatment of uterine and fetoplacental arteries with bFGF, VEGF, and EGF would further enhance the pregnancy-induced increase in PGI2 production. Duplicate uterine (UA) and fetoplacental (PA) artery (primary branch off of the umbilical cord = pPA; cotyledonary or tertiary = tPA) explants from seven late gestation sheep were placed in tissue culture (RPMI; 37 degrees C) for 24 h alone or with (1-100 ng/mL) bFGF, VEGF, or EGF. To evaluate the endothelial contribution to basal and stimulated PGI2 production and to determine whether it is de novo, arteries with and without endothelium from three additional late gestation ewes, tissues were incubated in the absence or presence of growth factors with or without meclofenamate (1 microM). The stable metabolite of PGI2, 6-keto-PGF1 alpha, was measured in culture media and expressed as ng/mg wet wt 24 h. PGI2 production by UA increased (p < 0.05) from 5.43 +/- 0.26 at control to 8.93 +/- 0.99 with 100 ng/mL bFGF. Although VEGF produced a similar response, EGF did not increase PGI2 production in UA. In pPA, 100 ng/mL bFGF induced a 2.2-fold increase (p < 0.01) in PGI2 production from 1.94 +/- 0.14 to 4.20 +/- 0.31; VEGF and EGF were without effect. In tPA, 50 and 100 ng/mL bFGF increased PGI2 production from 1.98 +/- 0.14 to 3.5 +/- 1.05 and 3.96 +/- 0.46 (p < 0.02). In tPA, VEGF did not increase PGI2 production; however, 10, 50, and 100 ng/mL EGF, enhanced (p < 0.03) PGI2 production from 1.98 +/- 0.14 to 3.39 +/- 0.62, 3.62 +/- 0.26, and 2.93 +/- 0.20. Endothelium removal and meclofenamate treatment caused a 90% and 100% decrease, respectively, in basal PGI2 production, with no recovery after treatment with growth factors. We conclude that PGI2 production is augmented by bFGF in UA, pPA and tPA, by VEGF in UA, and by EGF in tPA during ovine pregnancy. Basal and stimulated PGI2 secretion is endothelium-derived via de novo synthesis. bFGF, VEGF, and EGF, in addition to angiogenesis, may modulate PGI2 production, further enhancing blood flow to the growing uteroplacental bed.

Animals↗
PubMed1999

Basic FGF decreases clearance receptor of natriuretic peptides in fetoplacental artery endothelium.

Atrial natriuretic peptide (ANP) is present in the fetoplacental circulation of humans and sheep. The ANP-A receptor is the specific membrane receptor for ANP, which produces cGMP. The clearance receptor of natriuretic peptide (CR) is postulated to modulate local concentrations of ANP, thereby modulating cGMP production through the ANP-A receptor. Recently we reported that fetoplacental basic fibroblast growth factor (bFGF) and cGMP levels are increased dramatically during the third trimester of ovine gestation. Therefore we hypothesized that bFGF will downregulate CR expression in cultured ovine fetoplacental artery endothelial (OFPAE) cells via the mitogen-activated protein kinase (MAPK) signal cascade mechanism, thereby causing augmentation of ANP-mediated cGMP production. Western analysis and/or RT-PCR of CR expression were performed after treatment of OFPAE cells with bFGF (10 pg/ml-1 microgram/ml) with or without 50 microM PD-98059, a selective inhibitor of MAPK kinase. To investigate the possible effects of CR downregulation on the functional modulation of ANP-A receptor activation, cGMP production (20 min) by OFPAE cells was measured in response to ANP (10 pM-1 microM) with or without pretreatment (24 h) of 10 ng/ml bFGF. CR expression in OFPAE cells was dose dependently downregulated by 1-10 ng/ml bFGF treatment (maximum -69%), which was completely reversed by pretreatment with PD-98059. Treatment of OFPAE cells with 10 ng/ml bFGF (24 h) did not alter maximum ANP-A activity (cGMP production/20 min), but decreased the apparent ED(50) of ANP to stimulate cGMP production from 2.5 to 0.83 nM, suggesting the possibility that bFGF-mediated downregulation of CR may elevate ANP-mediated cGMP production responses. Thus bFGF downregulates CR mRNA and protein expressions via the MAPK cascade in OFPAE cells.

Animals↗
PubMed1999

Activation of the mitogen-activated protein kinase cascade is necessary but not sufficient for basic fibroblast growth factor- and epidermal growth factor-stimulated expression of endothelial nitric oxide synthase in ovine fetoplacental artery endothelial cells.

Basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) may play important roles in the placental vasculature, not only by controlling cell growth and differentiation, but also by mediating production of local vasodilators such as nitric oxide. As the mitogen-activated protein kinase (MAPK) signal cascade has been widely associated with cell growth in response to growth factors, herein we investigate whether bFGF, EGF, and VEGF also stimulate expression of endothelial nitric oxide synthase (eNOS) via activation of the MAPK cascade in ovine fetoplacental artery endothelial cells. The presence of the receptors for all three growth factors was confirmed by both immunocytochemistry and a functional cell proliferation assay. All three growth factors at 10 ng/ml rapidly (<10 min) activated MAPK. This activation was inhibited by PD 98059, a specific MAPK kinase inhibitor. bFGF and EGF, but not VEGF, dose- and time-dependently increased eNOS protein levels. Maximal stimulatory effects of bFGF and EGF on eNOS protein expression were observed at 10 ng/ml for 24 h of treatment and were associated with elevated eNOS messenger RNA. PD 98059 also significantly inhibited bFGF- and EGF-induced increases in eNOS protein expression. Because treatment with all three growth factors resulted in activation of the MAPK cascade, while bFGF and EGF, but not VEGF, increased eNOS expression, we conclude that activation of the MAPK cascade is necessary, but not sufficient, for bFGF- and EGF-induced increases in eNOS protein expression in ovine fetoplacental artery endothelial cells. Thus, additional signaling pathways are implicated in the different controls of eNOS expression and mitogenesis by growth factors.

Animals↗
PubMed1998

Specific pregnancy-induced angiotensin II type-1 receptor expression in ovine uterine artery does not involve formation of alternate splice variants or alternate promoter usage.

Recently we reported that pregnancy is associated with a dramatic increase in angiotensin II type-1 receptor (AT1-R; both protein and mRNA) in ovine uterine artery endothelial cells (UAEC), which far exceeds that seen in omental (systemic) arteries. Recent reports also suggest that alternate splicing of AT1-R mRNA may play a role in regulation of AT1-R expression in humans. Herein, we have investigated the possibility of alternate transcript splicing/promoter usage in UAEC from pregnant ewes by 5'-RACE (rapid amplification of cDNA 5'-ends). To provide our control "reference" sequences, we first performed 5'-RACE analysis of AT1-R mRNA transcripts in liver, kidney, and adrenal cortex. Analysis of 17 resultant clones showed exceptional homology, indicating that a single identically spliced mRNA product is observed in all three ovine tissues. Homology of the 5'-untranslated region to that of the human was low (34.2%), but four in-context start/stop codons and the beginning of human exons 1 and 5 were highly conserved. Subsequently we isolated 30 individual clones using UAEC RNA from three pregnant ewes and found no evidence of any sequence formed through unique splicing or promoter usage. We conclude that the pregnancy-induced increase in AT1-R expression unique to UAEC during pregnancy is not mediated by splicing of a unique transcript or unique promoter usage.

Angiotensin II↗