Vascular adaptation in pregnancy and endothelial dysfunction in preeclampsia.

Maternal vascular adaptation to pregnancy is critically important to expand the capacity for blood flow through the uteroplacental unit to meet the needs of the developing fetus. Failure of the maternal vasculature to properly adapt can result in hypertensive disorders of pregnancy such as preeclampsia (PE). Herein, we review the endocrinology of maternal adaptation to pregnancy and contrast this with that of PE. Our focus is specifically on those hormones that directly influence endothelial cell function and dysfunction, as endothelial cell dysfunction is a hallmark of PE. A variety of growth factors and cytokines are present in normal vascular adaptation to pregnancy. However, they have also been shown to be circulating at abnormal levels in PE pregnancies. Many of these factors promote endothelial dysfunction when present at abnormal levels by acutely inhibiting key Ca2+ signaling events and chronically promoting the breakdown of endothelial cell-cell contacts. Increasingly, our understanding of how the contributions of the placenta, immune cells, and the endothelium itself promote the endocrine milieu of PE is becoming clearer. We then describe in detail how the complex endocrine environment of PE affects endothelial cell function, why this has contributed to the difficulty in fully understanding and treating this disorder, and how a focus on signaling convergence points of many hormones may be a more successful treatment strategy.

Metabolic gene profile in early human fetal heart development.

The primitive cardiac tube starts beating 6-8 weeks post fertilization in the developing embryo. In order to describe normal cardiac development during late first and early second trimester in human fetuses this study used microarray and pathways analysis and created a corresponding 'normal' database. Fourteen fetal hearts from human fetuses between 10 and 18 weeks of gestational age (GA) were prospectively collected at the time of elective termination of pregnancy. RNA from recovered tissues was used for transcriptome analysis with Affymetrix 1.0 ST microarray chip. From the amassed data we investigated differences in cardiac development within the 10-18 GA period dividing the sample by GA in three groups: 10-12 (H1), 13-15 (H2) and 16-18 (H3) weeks. A fold change of 2 or above adjusted for a false discovery rate of 5% was used as initial cutoff to determine differential gene expression for individual genes. Test for enrichment to identify functional groups was carried out using the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Array analysis correctly identified the cardiac specific genes, and transcripts reported to be differentially expressed were confirmed by qRT-PCR. Single transcript and Ontology analysis showed first trimester heart expression of myosin-related genes to be up-regulated >5-fold compared with second trimester heart. In contrast the second trimester hearts showed further gestation-related increases in many genes involved in energy production and cardiac remodeling. In conclusion, fetal heart development during the first trimester was dominated by heart-specific genes coding for myocardial development and differentiation. During the second trimester, transcripts related to energy generation and cardiomyocyte communication for contractile coordination/proliferation were more dominant. Transcripts related to fatty acid metabolism can be seen as early as 10 weeks and clearly increase as the heart matures. Retinol receptor and gamma-aminobutyric acid (GABA) receptor transcripts were detected, and have not been described previously in human fetal heart during this period. For the first time global gene expression of heart has been described in human samples to create a database of normal development to understand and compare with known abnormal fetal heart development.

Differential changes in gene expression in human brain during late first trimester and early second trimester of pregnancy.

OBJECTIVE: This study aimed to describe brain development during the first (B1) and second trimester (B3) in human fetuses. DESIGN: Ten brains from 10 to 18 weeks of gestational age (GA) were collected, and the RNA was used for transcriptome analysis (Affymetrix 1.0 ST microarray chip). Differences in brain development within 10 to 18 GA were investigated by dividing the sample into 10 to 12 (B1), 13 to 15(B2) and 16 to 18(B3) weeks. A fold change of 2 or above, with a false discovery rate of 5%, was used as cut-off to determine differential gene expression for individual genes. Quantitative real-time PCR was used to confirm differences. Tests for enrichment procedures (using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes) were then used to identify functional groups of mRNA. RESULTS: At 10 to 12 weeks, brains showed neuronal migration to be upregulated. From 10 to 18 weeks, brains showed genes coding for neuronal migration, differentiation and connectivity upregulated. ALDH1A1 and NPY genes, marker of spinal cord and striatum, were upregulated in B1 and B3 brains, respectively. Also, SLITRK6-HAS2 and CRYAB-PCDH18 genes for ear and eye sensory input were upregulated in B1. CONCLUSIONS: For the first time, brain global gene expression was described in human samples. Period B1 was dominated by genes coding for neuronal migration, differentiation, programmed cell death and sensory organs. B3 was dominated by neuronal proliferation, branching and myelination. Creating such a database will allow comparison with abnormals in future studies.

Pregnancy, programming and preeclampsia: gap junctions at the nexus of pregnancy-induced adaptation of endothelial function and endothelial adaptive failure in PE.

The challenge of pregnancy to the mother requires that her own metabolic and endocrine needs be met while also taking on the literally growing demands of the unborn child. While all of the mother's organs require continued support, the uterus and now added placenta must also develop substantially. One critical area of adaptation is thus the ability to provide added blood flow over and above that already serving the preexisting maternal organs. Previous reviews have covered in detail how this is achieved from an endocrine or indeed vascular physiology standpoint and we will not repeat that here. Suffice it to say in addition to new vessel growth, there is also the need to achieve reduced vascular resistance through maintenance of endothelial vasodilation, particularly through NO and PGI2 production in response to multiple agonists and their associated cell signaling systems. In this review, we continue our focus on pregnancy adaptive changes at the level of cell signaling, with a particular emphasis now on the developing story of the critical role of gap junctions. Remapping of cell signaling itself beyond changes in individual hormones and respective receptors brings about global changes in cell function, and recent studies have revealed that such post-receptor changes in cell signaling are equally if not more important in the process of pregnancy adaptation of endothelial function than the upregulated expression of vasodilator synthetic pathways themselves. The principle significance, however, of reviewing this aspect of pregnancy adaptation of endothelial cell function is that these same gap junction proteins that mediate pregnancy-adapted changes in vasodilatory signaling function may also be the focal point of failure in diseased pregnancy, and clues as to how and why are given by comparing studies of Cx43 functional suppression at wound sites with studies of preeclamptic pregnancy. If preeclamptic pregnancy is indeed a pregnancy misconstrued by the body in endocrine terms to be a wound, then the kinases so activated that correspondingly suppress Cx43 function in the vascular endothelium may also be valid pharmacologic targets for novel therapies in the near future.

eNOS activation and NO function: pregnancy adaptive programming of capacitative entry responses alters nitric oxide (NO) output in vascular endothelium--new insights into eNOS regulation through adaptive cell signaling.

In pregnancy, vascular nitric oxide (NO) production is increased in the systemic and more so in the uterine vasculature, thereby supporting maximal perfusion of the uterus. This high level of functionality is matched in the umbilical vein, and in corresponding disease states such as pre-eclampsia, reduced vascular responses are seen in both uterine artery and umbilical vein. In any endothelial cell, NO actually produced by endothelial NO synthase (eNOS) is determined by the maximum capacity of the cell (eNOS expression levels), eNOS phosphorylation state, and the intracellular [Ca(2+)](i) concentration in response to circulating hormones or physical forces. Herein, we discuss how pregnancy-specific reprogramming of NO output is determined as much by pregnancy adaptation of [Ca(2+)](i) signaling responses as it is by eNOS expression and phosphorylation. By examining the changes in [Ca(2+)](i) signaling responses from human hand vein endothelial cells, uterine artery endothelial cells, and human umbilical vein endothelial cells in (where appropriate) nonpregnant, normal pregnant, and pathological pregnant (pre-eclamptic) state, it is clear that pregnancy adaptation of NO output occurs at the level of sustained phase 'capacitative entry' [Ca(2+)](i) response, and the adapted response is lacking in pre-eclamptic pregnancies. Moreover, gap junction function is an essential permissive regulator of the capacitative response and impairment of NO output results from any inhibitor of gap junction function, or capacitative entry using TRPC channels. Identifying these [Ca(2+)](i) signaling mechanisms underlying normal pregnancy adaptation of NO output not only provides novel targets for future treatment of diseases of pregnancy but may also apply to other common forms of hypertension.

Testosterone restores respiratory long term facilitation in old male rats by an aromatase-dependent mechanism.

Steroidal sex hormones play an important role in the neural control of breathing. Previous studies in our laboratory have shown that gonadectomy in young male rats (3 months) eliminates a form of respiratory plasticity induced by intermittent hypoxia, known as long term facilitation (LTF). Testosterone replenishment restores LTF in gonadectomized male rats, and this is dependent on the conversion of testosterone to oestradiol by aromatase. By middle age (12 months), male rats no longer exhibit LTF of hypoglossal motor output; phrenic LTF is significantly reduced, and this persists into old age. We tested the hypothesis that LTF can be restored in old male rats by administration of testosterone. Intact Fischer 344 rats (>20 months) were implanted with Silastic tubing containing testosterone (T), T plus an aromatase inhibitor (T+ADT), or 5α-dihydrotestosterone (DHT), a form of testosterone not converted to oestradiol. One week post-surgery, LTF of hypoglossal and phrenic motor output was measured. By comparison with control rats, hypoglossal LTF was increased in testosterone-treated rats, with levels approaching that of normal young rats. LTF was not restored in T+ADT or DHT-treated rats. Aromatase levels in hypoglossal and phrenic nuclei did not change with age. As serum testosterone levels did not decline with age, local bioavailability of testosterone in old rats may be a limiting factor in the expression of this form of respiratory plasticity. Our findings suggest that testosterone supplementation could potentially be used to enhance upper airway control in the elderly.

Nonhuman primates as models for human adrenal androgen production: function and dysfunction.

The origin of circulating DHEA and adrenal-derived androgens in humans and nonhuman primates is largely distinct from other mammalian species. In humans and many Old world primates, the fetal adrenal gland and adult zona reticularis (ZR) are known to be the source for production of DHEA (and DHEAS) in mg quantities. In spite of similarities there are also some differences. Herein, we take a comparative endocrine approach to the diversity of adrenal androgen biosynthesis and its developmental timing in three primate species to illustrate how understanding such differences may provide unique insight into mechanisms underlying adrenal androgen regulation and its pathophysiology in humans. We contrast the conventional developmental onset of adrenal DHEA biosynthesis at adrenarche in humans with (1) an earlier, peri-partutrition onset of adrenal DHEA synthesis in rhesus macaques (Old World primate) and (2) a more dynamic and reversible onset of adrenal DHEA biosynthesis in female marmosets (New World primate), and further consider these events in terms of the corresponding developmental changes in expression of CYP17, HSD3B2 and CYB5 in the ZR. We also integrate these observations with recently described biochemical characterization of CYP17 cDNA cloned from each of these nonhuman primate species and the corresponding effects of phosphorylation versus CYB5 coexpression on 17,20 lyase versus 17-hydroxylase activity in each case. In addition, female rhesus macaques exposed in utero to exogenous androgen excess, exhibit symptoms of adrenal hyperandrogenism in adult females in a manner reminiscent of that seen in the human condition of PCOS. The possible mechanisms underlying such adrenal hyperandrogenism are further considered in terms of the effects of altered relative expression of CYP17, HSD3B2 and CYB5 as well as the altered signaling responses of various kinases including protein kinase A, or the insulin sensitive PI3-kinase/AKT signaling pathway which may impact on 17,20 lyase activity. We conclude that while the triggers for the onset of ZR function in all three species show clear differences (age, stage of development, social status, gender), there are still common mechanisms driving an increase in DHEA biosynthesis in each case. A full understanding of the mechanisms that control 17,20 lyase function and dysfunction in humans may best be achieved by comparative studies of the endocrine mechanisms controlling adrenal ZR function and dysfunction in these nonhuman primate species.

Structural characterization of the interaction of mTOR with phosphatidic acid and a novel class of inhibitor: compelling evidence for a central role of the FRB domain in small molecule-mediated regulation of mTOR.

The mammalian target of rapamycin (mTOR) is a large, multidomain protein kinase, which plays a central role in the regulation of cell growth and has recently emerged as an essential target of survival signals in many types of human cancer cells. Here, we report the solution structures of complexes formed between the FKBP12-rapamycin binding (FRB) domain of mTOR and phosphatidic acid, an important cellular activator of the kinase, and between the FRB domain and a novel inhibitor (HTS-1). The overall structure of the FRB domain is very similar to that seen in the ternary complex formed with FKBP12 and the immunosuppressive drug rapamycin; however, there are significant changes within the rapamycin-binding site with important consequences for rational drug design. The surface of the FRB domain contains a number of distinctive features that have previously escaped attention, including a potential new regulatory site on the opposite face to that involved in the binding of rapamycin, which displays the features expected for a specific binding site for a small molecule. The interaction sites for phosphatidic acid and HTS-1 were found to closely match the site responsible for rapamycin binding. In addition, the structures determined for the FRB-phosphatidic acid and FRB-HTS-1 complexes revealed a striking similarity between the conformations of buried portions of the ligands and that seen for the rapamycin backbone in contact with the domain. Our findings further highlight the importance of the FRB domain in small molecule-mediated regulation of mTOR, demonstrate the ability to identify novel inhibitors of mTOR that bind tightly to the rapamycin-binding site in the absence of FKBP12, and identify a potential new regulatory site that may be exploited in the design of new anticancer drugs.

Functional characterization of HUVEC-CS: Ca2+ signaling, ERK 1/2 activation, mitogenesis and vasodilator production.

While many endothelial cell lines exist, few are of human origin with characteristics close to the parent endothelial cell. We derived a subline (HUVEC-CS) of immortalized human umbilical vein endothelial cells (HUVEC-C) that proliferate in standard growth media and exhibit positive acetylated low-density lipoprotein (AcLDL) uptake, express eNOS, CD31 and ve-cadherin, and spontaneously form capillary-like structures when grown on Matrigel. HUVEC-CS also maintain endothelial cell characteristics at the level of mitogenesis, kinase activation and vasodilator production. Like primary HUVEC cells, HUVEC-CS express many of the key proteins necessary for vasodilator production, including epithelial nitric oxide synthase (eNOS), HSP 90, cav-1 and -2, cPLA2, and COX-1 and -2. Prostaglandin I synthase (PGIS) was not detectable by Western blot analysis, consistent with primary HUVEC in which PGI2 production is minimal. Receptors were detected for angiotensin II (AII), bradykinin, ATP and growth factors. ATP induced a dose- and time-dependent rise in the intracellular free Ca2+ concentration ([Ca2+]i). Initially, ATP stimulates P2Y receptors rather than P2X receptors, as demonstrated by the inability of ATP to initiate a Ca2+ response subsequent to emptying of the internal Ca2+ stores by thapsigargin. AII, bradykinin, epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) also caused a rise in [Ca2+]i in a subset of the cells. ATP, basic fibroblastic growth factor (bFGF), EGF and VEGF induced mitogenesis and caused a rise in ERK 2 activation within 10 min. L-Arginine to L-citrulline conversion assays showed that ATP, EGF and VEGF induced a significant rise in eNOS activity, and this correlates with an ability to induce Ca2+ mobilization and ERK 2 activation. In conclusion, HUVEC-CS are indeed endothelial cells and appear to be functionally very similar to primary HUVEC. These cells will prove a valuable tool for future studies in both basic and therapeutic sciences.

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.

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).

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.

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.

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.

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.

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.

Laparoscopic cholecystectomy does not demonstrably decrease survival of patients with serendipitously treated gallbladder cancer.

BACKGROUND: The purpose of this study was to evaluate the possibility that laparoscopic cholecystectomy has worsened the prognosis of patients with resected gallbladder cancer; particularly for patients whose cancer was accidentally resected. STUDY DESIGN: We conducted a retrospective review of Connecticut Tumor Registry data and data extracted from individual patient records at 15 of 30 hospitals in Connecticut reporting data to the Registry, at two separate time points, 1985-1988 (immediate prelaparoscopic era) and 1992-95 (laparoscopic cholecystectomy well established). There were 194 and 208 patients in each 3-year period, respectively. Additional information was extracted from hospital records in 82 and 91 patients, respectively. Twenty-five percent of patients in both data sets presented with "local" or Tis, T1, T2 disease. RESULTS: Three-year survival for localized disease was 29% in the prelaparoscopic period and 34% once laparoscopic cholecystectomy was established. But analysis of individual patient records indicated that 36% of patients from the laparoscopic period did not actually undergo a laparoscopic procedure. Fifty-nine patients had their gallbladder cancer discovered in the specimen postoperatively (serendipitously treated). A higher proportion of cancers were discovered postoperatively in the laparoscopic era (44% versus 24%). Three-year survival for these patients was 25%. If the data from the two eras are grouped according to whether or not the cancer-bearing gallbladder was manipulated laparoscopically, 24 of 59 patients (41%) turned out to be at risk for the possibility of increased laparoscopic dissemination of tumor. Survival of these patients (11-month median survival) was not statistically different from survival of patients whose serendipitously discovered gallbladder cancer was never manipulated laparoscopically (16-month median survival); p = 0.54 by log rank test. CONCLUSIONS: The widespread adoption of laparoscopic cholecystectomy did not worsen the survival of patients with gallbladder cancer, and patients with serendipitously treated gallbladder cancers did not have a worse survival after laparoscopic manipulation than after a standard open cholecystectomy. The laparoscopic aspects of operative manipulation of a gallbladder with cancer in it do not appear to be a proximate cause of the poor prognosis in this disease.

Antenatal steroid treatment and adverse fetal effects: what is the evidence?

This article reviews current animal and human data regarding possible adverse fetal effects from antenatal steroid treatment. Although it is now well accepted that such treatment is of benefit to fetal lung development, the potential for adverse fetal outcomes as a result of single or multiple glucocorticoid dosing has not been widely recognized. There are now growing concerns, based on animal and some human data, that repeated antenatal doses could lead to a decrease in birth weight, a decrease in fetal brain and other organ size, and abnormal neuronal development. Previous investigations have been hampered by nonstandardization in the type of glucocorticoid, route of delivery, timing of administration, and number of treatment courses. It is recommended that these concerns be addressed through large randomized, controlled clinical trials. In the meantime, it would be prudent to minimize antenatal steroid treatments to a single course with repeated dosing only if there is a persistent threat of preterm delivery. The practice of giving weekly injections of steroids starting at fetal viability and continuing into the third trimester is not supported.