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.     

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.     

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.     

Double-label expression studies of prostacyclin synthase, thromboxane synthase and COX isoforms in normal aortic endothelium

We have performed double-label immunofluorescence microscopy studies to evaluate the extent of co-localization of prostacyclin synthase (PGIS) and thromboxane synthase (TXS) with cyclooxygenase (COX)-1 and COX-2 in normal aortic endothelium. In dogs, COX-2 expression was found to be restricted to small foci of endothelial cells while COX-1, PGIS and TXS were widely distributed throughout the endothelium. Quantification of the total cross-sectioned aortic endothelium revealed a 6- to 7-fold greater expression of COX-1 relative to COX-2 (55 vs. 8%) and greater co-distribution of PGIS with COX-1 compared to COX-2 (19 vs. 3%). These results are in contrast to the extensive co-localization of PGIS and COX-2 in bronchiolar epithelium. In rat and human aortas, immunofluorescence studies also showed significant COX-1 and PGIS co-localization in the endothelium. Only minor focal COX-2 expression was detected in rat endothelium, similar to the dog, while COX-2 was not detected in human specimens. Inhibition studies in rats showed that selective COX-1 inhibition caused a marked reduction of 6-keto-PGF(1alpha) and TXB(2) aortic tissue levels, while COX-2 inhibition had no significant effect, providing further evidence for a functionally larger contribution of COX-1 to the synthesis of prostacyclin and thromboxane in aortic tissue. The data suggest a major role for COX-1 in the production of both prostacyclin and thromboxane in normal aortic tissue. The extensive co-localization of PGIS and COX-2 in the lung also indicates significant tissue differences in the co-expression patterns of these two enzymes.     

Possible mechanisms underlying pregnancy-induced changes in uterine artery endothelial function

The last 10 years has seen a dramatic increase in our understanding of the mechanisms underlying the pregnancy-specific adaptation in cardiovascular function in general and the dramatic changes that occur in uterine artery endothelium in particular to support the growing fetus. The importance of these changes is clear from a number of studies linking restriction of uterine blood flow (UBF) and/or endothelial dysfunction and clinical conditions such as intrauterine growth retardation (IUGR) and/or preeclampsia in both humans and animal models; these topics are covered only briefly here. The recent developments that prompts this review are twofold. The first is advances in an understanding of the cell signaling processes that regulate endothelial nitric oxide synthase (eNOS) in particular (Govers R and Rabelink TJ. Am J Physiol Renal Physiol 280: F193-F206, 2001). The second is the emerging picture that uterine artery (UA) endothelial cell production of nitric oxide (NO) as well as prostacyclin (PGI2) may be as much a consequence of cellular reprogramming at the level of cell signaling as due to tonic stimuli inducing changes in the level of expression of eNOS or the enzymes of the PGI2 biosynthetic pathway (cPLA2, COX-1, PGIS). In reviewing just how we came to this conclusion and outlining the implications of such a finding, we draw mostly on data from ovine or human studies, with reference to other species only where directly relevant.     

Cyclooxygenase-2-derived endogenous prostacyclin reduces apoptosis and enhances embryo viability in mouse

The role of prostaglandins (PGs) in apoptosis in preimplantation mice embryo development is reported in this study. It is known that apoptosis plays a very important role in normal mice embryo development. Very few reports are available on this subject. Embryos (6-8 cells) were cultured in the presence of a selective cyclooxygenase (COX)1 inhibitor (SC560), a selective COX2 inhibitor (NS398) and a selective prostacyclin synthase (PGIS) inhibitor (U51605) in a 48-h culture. In another experiment, culture media were supplemented with prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2 or prostacyclin) analogues. The apoptosis was evaluated by detection of active caspase-3. It was strongly detected in the presence of selective COX-2 and PGIS inhibitors, which can be decreased by a PGI2 analogue. In our embryo transfer experiment, the implantation rate decreased with exposure to either the COX2 or the PGIS inhibitor which is increased further after PGI2 supplementation. The level of PGI2 is also higher at the 8-16-cell stage, compaction and blastocyst stage than PGE2. All these results indicate that COX2-derived PGI2 plays an important role in preimplantation embryo development and acts as an antiapopetic factor in in vitro culture.     

Estrogen potentiates constrictor prostanoid function in female rat aorta by upregulation of cyclooxygenase-2 and thromboxane pathway expression

Estrogen potentiates vascular reactivity to vasopressin (VP) by enhancing constrictor prostanoid function. To determine the cellular and molecular mechanisms, the effects of estrogen on arachidonic acid metabolism and on the expression of constrictor prostanoid pathway enzymes and endoperoxide/thromboxane receptor (TP) were determined in the female rat aorta. The release of thromboxane A2 (TxA2) and prostacyclin (PGI2) was measured in male (M), intact-female (Int-F), ovariectomized-female (OvX-F), and OvX + 17beta-estradiol-replaced female (OvX + ER-F) rats. The expression of mRNA for cyclooxygenase (COX)-1, COX-2, thromboxane synthase (TxS), and TP by aortic endothelium (Endo) and vascular smooth muscle (VSM) of these four experimental groups was measured by RT-PCR. The expression of COX-1, COX-2, and TxS proteins by Endo and VSM was also estimated by immunohistochemistry (IHC). Basal release of TxA2 and PGI2 was similar in M (18.8 +/- 1.9 and 1,723 +/- 153 pg/mg ring wt/45 min, respectively) and Int-F (20.2 +/- 4.2 and 1,488 +/- 123 pg, respectively) rat aortas. VP stimulated the dose-dependent release of TxA2 and PGI2 from both male and female rat aorta. OvX markedly attenuated and ER therapy restored VP-stimulated release of TxA2 and PGI2 in female rats. No differences in COX-1 mRNA levels were detected in either Endo or VSM of the four experimental groups (P > 0.1). The expression of both COX-2 and TxS mRNA were significantly higher (P < 0.05) in both Endo and VSM of Int-F and OvX + ER-F, compared with M or OvX-F. Expression of TP mRNA was significantly higher in VSM of Int-F and OvX + ER-F compared with M or OvX-F. IHC revealed the uniform staining of COX-1 in VSM of the four experimental groups, whereas staining of COX-2 and TxS was greater in Endo and VSM of Int-F and OvX + ER-F than in OvX-F or M rats. These data reveal that estrogen enhances constrictor prostanoid function in female rat aorta by upregulating the expression of COX-2 and TxS in both Endo and VSM and by upregulating the expression of TP in VSM.     

Inducible COX-2 dominates over COX-1 in prostacyclin biosynthesis: mechanisms of COX-2 inhibitor risk to heart disease

AimOur aim is to understand the molecular mechanisms of the selective nonsteroidal anti-inflammatory drugs (NSAID), cyclooxygenase-2 (COX-2) inhibitors', higher “priority” to reduce synthesis of the vascular protector, prostacyclin (PGI2), compared to that of nonselective NSAIDs.Main methodsCOX-1 or COX-2 was co-expressed with PGI2 synthase (PGIS) in COS-7 cells. The Km and initial velocity (½t Vmax) of the coupling reaction between COX-1 and COX-2 to PGIS were established. The experiment was further confirmed by a kinetics study using hybrid enzymes linking COX-1 or COX-2 to PGIS. Finally, COX-1 or COX-2 and PGIS were respectively fused to red (RFP) and cyanic (CFP) fluorescence proteins, and co-expressed in cells. The distances between COXs and PGIS were compared by FRET.Key findingsThe Km for converting arachidonic acid (AA) to PGI2 by COX-2 coupled to PGIS is ~ 2.0 μM; however, it was 3-fold more (~ 6.0 μM) for COX-1 coupled to PGIS. The Km and ½t Vmax for COX-2 linked to PGIS were ~ 2.0 μM and 20 s, respectively, which were 2–5 folds faster than that of COX-1 linked to PGIS. The FRET study found that the distance between COX-2-RFP and PGIS–CFP is shorter than that between COX-1-RFP and PGIS–CFP.SignificanceThe study provided strong evidence suggesting that the low Km, faster ½t Vmax, and closer distance are the basis for COX-2 dominance over COX-1 (coupled to PGIS) in PGI2 synthesis, and further demonstrated the mechanisms of selective COX-2 inhibitors with higher potential to reduce synthesis of the vascular protector, PGI2.     

Characterization of the PGI2/IP system in cultured rat mesangial cells

Mesangial cells play an important role in glomerular function. They are an important source of cyclooxygenase (COX)-derived arachidonic acid metabolites, including prostaglandin E(2) and prostacyclin. Prostacyclin receptor (IP) mRNA was amplified from cultured mesangial cell total RNA by RT-PCR. While the prostaglandin E(2) receptor subtype EP(2) was not detected, EP(1,3,4) mRNA was amplified. Also, IP protein was noted in mesangial cells, proximal tubules, inner medullary collecting ducts, and the inner and outer medulla. But no protein was detected in whole cortex preparations. Prostacyclin analogues: cicaprost and iloprost, increased cAMP levels in mesangial cells. On the other hand, arginine-vasopressin and angiotensin II increased intracellular calcium in mesangial cells, but cicaprost, iloprost and prostaglandin E(2) had no effect. Moreover, a 50% inhibition of cicaprost- and iloprost-cAMP stimulation was observed upon mesangial cell exposure to 25 and 35 mM glucose for 5 days. But no change in IP mRNA was observed at any glucose concentration or time exposure. Although 25 mM glucose had no effect on COX-1 protein levels, COX-2 was increased up to 50%. In contrast, PGIS levels were reduced by 50%. Thus, we conclude that the prostacyclin/IP system is present in cultured rat mesangial cells, coupling to a cAMP stimulatory pathway. High glucose altered both enzymes in the PGI(2) synthesis pathway, increasing COX-2 but reducing PGIS. In addition, glucose diminished the cAMP response to prostacyclin analogues. Therefore, glucose attenuates the PGI(2)/IP system in cultured rat mesangial cells.     

Mechanosensing role of caveolae and caveolar constituents in human endothelial cells

A variety of evidence suggests that endothelial cell functions are impaired in altered gravity conditions. Nevertheless, the effects of hypergravity on endothelial cell physiology remain unclear. In this study we cultured primary human endothelial cells under mild hypergravity conditions for 24-48 h, then we evaluated the changes in cell cycle progression, caveolin1 gene expression and in the caveolae status by confocal microscopy. Moreover, we analyzed the activity of enzymes known to be resident in caveolae such as endothelial nitric oxide synthase (eNOS), cycloxygenase 2 (COX-2), and prostacyclin synthase (PGIS). Finally, we performed a three-dimensional in vitro collagen gel test to evaluate the modification of the angiogenic responses. Results indicate that hypergravity shifts endothelial cells to G(0)/G(1) phase of cell cycle, reducing S phase, increasing caveolin1 gene expression and causing an increased distribution of caveolae in the cell interior. Hypergravity also increases COX-2 expression, nitric oxide (NO) and prostacyclin (PGI2) production, and inhibits angiogenesis as evaluated by 3-D collagen gel test, through a pathway not involving apoptosis. Thus, endothelial cell caveolae may be responsible for adaptation of endothelium to hypergravity and the mechanism of adaptation involves an increased caveolin1 gene expression coupled to upregulation of vasodilators as NO and PGI2.     

Selective inhibition of prostacyclin synthase activity by rofecoxib

The development of cyclooxygenase-2 (COX-2) selective inhibitors prompted studies aimed at treating chronic inflammatory diseases and cancer by using this new generation of drugs.Yet, several recent reports pointed out that long-term treatment of patients with COX-2 selective inhibitors (especially rofecoxib) caused severe cardiovascular complicances. The aim of this study was to ascertain whether, in addition to inhibiting COX-2, rofecoxib may also affect prostacyclin (PGI2) level by inhibiting PGI2 forming enzyme (prostacyclin synthase, PGIS). In order to evaluate if selective (celecoxib, rofecoxib) and non-selective (aspirin, naproxen) anti-inflammatory compounds could decrease PGI2 production in endothelial cells by inhibiting PGIS, we analyzed the effect of anti-inflammatory compounds on the enzyme activity by ELISA assay after addition of exogenous substrate, on PGIS protein levels by Western blotting and on its subcellular distribution by confocal microscopy. We also analyzed the effect of rofecoxib on PGIS activity in bovine aortic microsomal fractions enriched in PGIS. This study demonstrates an inhibitory effect of rofecoxib on PGIS activity in human umbilical vein endothelial (HUVE) cells and in PGIS-enriched bovine aortic microsomal fractions, which is not observed by using other anti-inflammatory compounds. The inhibitory effect of rofecoxib is associated neither to a decrease of PGIS protein levels nor to an impairment of the enzyme intracellular localization. The results of this study may explain the absence of a clear relationship between COX-2 selectivity and cardiovascular side effects. Moreover, in the light of these results we propose that novel selective COX-2 inhibitors should be tested on PGI2 synthase activity inhibition.     

Peroxynitrite and protein tyrosine nitration of prostacyclin synthase

When working on the regulation of prostacyclin synthase (PGIS), we found that PGIS was selectively inhibited by peroxynitrite (ONOO-), a potent oxidant formed by the combination of superoxide anion and nitric oxide (NO) at a rate of diffusion-controlled. None of the cellular antioxidants studied (i.e. GSH, Vitamins C and E, and others) prevented the inhibition of ONOO- on PGIS. This unexpected behavior was explained by a catalytic reaction of the iron-thiolate center of PGIS with ONOO- anion. In contrast, ONOO- activated both thromboxane A2-synthase and cyclooxygenases. In addition, we demonstrated that sub-micromolar levels of ONOO- inhibited PGI2-dependent vasorelaxation and triggered a PGH2-dependent vasospasm, indicating that ONOO- increased PGH2 formation as a consequence of PGIS nitration. We have subsequently demonstrated that endogenous ONOO- caused PGIS nitration and TxA2 activation in several diseased conditions such as atherosclerotic vessels, hypoxia-reperfusion injury, cytokines-treated cells, diabetes, as well as hypertension. Since NO is produced physiologically it seems that excessive formation of superoxide not only eliminates the vasodilatory, growth-inhibiting, anti-thrombotic and anti-adhesive effects of NO and PGI2 but also allows and promotes an action of the potent vasoconstrictor, prothrombotic agent, growth promoter, and leukocyte adherer, PGH2. We conclude that the nitration of PGIS nitration might be a new pathogenic mechanism for superoxide-induced endothelium dysfunction often observed in vascular diseases such as atherosclerosis, hypertension, ischemia, endotoxic shock, and diabetes.     

Histamine directly and synergistically with lipopolysaccharide stimulates cyclooxygenase-2 expression and prostaglandin I(2) and E(2) production in human coronary artery endothelial cells

Although histamine plays an essential role in inflammation, its influence on cyclooxygenases (COX) and prostanoid homeostasis is not well understood. In this study, we investigated the effects of histamine on the expression of COX-1 and COX-2 and determined their contribution to the production of PGE(2), prostacyclin (PGI(2)), and thromboxane A(2) in human coronary artery endothelial cells (HCAEC). Incubation of HCAEC monolayers with histamine resulted in marked increases in the expression of COX-2 and production of PGI(2) and PGE(2) with no significant change in the expression of COX-1. Histamine-induced increases in PGI(2) and PGE(2) production were due to increased expression and function of COX-2 because gene silencing by small interfering RNA or inhibition of the catalytic activity by a COX-2 inhibitor blocked prostanoid production. The effects of histamine on COX-2 expression and prostanoid production were mediated through H(1) receptors. In addition to the direct effect, histamine was found to amplify LPS-stimulated COX-2 expression and PGE(2) and PGI(2) production. In contrast, histamine did not stimulate thromboxane A(2) production in resting or LPS-activated HCAEC. Histamine-induced increases in the production of PGE(2) and PGI(2) were associated with increased expression of mRNA encoding PGE(2) and PGI(2) synthases. The physiological role of histamine on the regulation of COX-2 expression in the vasculature is indicated by the findings that the expression of COX-2 mRNA, but not COX-1 mRNA, was markedly reduced in the aortic tissues of histidine decarboxylase null mice. Thus, histamine plays an important role in the regulation of COX-2 expression and prostanoid homeostasis in vascular endothelium.     

The influence of reproductive phase on lipopolysaccharide stimulation of prostacyclin production and cyclooxygenase-2 protein concentrations in reproductive and systemic arteries of the sheep

Cyclooxygenase, the enzyme that converts arachidonate to prostaglandins, plays a regulatory role in vasodilation under normal and pathological conditions. Studies were conducted to determine the effects of reproductive phase and lipopolysaccharide (LPS) on production of PGI2 and amounts of cyclooxygenase protein in uterine, mammary, mesenteric, and renal arteries. Arteries were collected from ewes during the follicular (Day 0 = estrus) or luteal (Day 10) phase of the estrous cycle and were cultured in the presence of LPS. After 24 h, media were collected and analyzed for 6-keto-PGF1alpha, the stable metabolite of PGI2. In addition, arteries were collected and homogenized and the relative concentration of cyclooxygenase was determined via Western analysis. Lipopolysaccharide stimulated PGI2 production in all four-artery types from both follicular and luteal phase ewes (p < 0.001). Upon LPS stimulation, uterine and mammary arteries produced more PGI2 compared to mesenteric and renal arteries (p = 0.04). The phase of estrous cycle did not affect PGI2 production by any of the artery populations exposed to LPS (p = 0.35). There was no cyclooxygenase-2 in untreated uterine and mammary arteries and no cyclooxygenase-2 was detected in untreated or LPS-treated mesenteric and renal arteries. In contrast, LPS-treated uterine and mammary arteries from luteal phase ewes had higher (p = 0.064) cyclooxygenase-2 concentrations than those from follicular phase ewes. These results suggest that the hormone conditions of the follicular (high estrogen) and luteal (high progesterone) phases of the ovarian cycle play a role in regulating uterine and mammary artery but not mesenteric and renal artery response to LPS.     

Production of prostacyclin and prostaglandin E2 in resting and IL-1beta-stimulated A549, HUVEC and hybrid EA.HY 926 cells.

Production of arachidonic acid (AA) metabolites - prostacyclin (PGI(2)) in large vessels and prostaglandin E(2) (PGE(2)) in microcirculation is intrinsically involved in maintenance of vascular wall homeostasis. EA.hy 926 is a hybrid cell line, is derived by fusion of HUVEC with A549 cells. The aim of this study was to examine the production of prostacyclin and PGE2 in resting and IL-1beta-stimulated EA.ha 926 cells, in comparison with its progenitor cells. Non-stimulated EA.hy 926 cells has been found to produce much lower amounts of prostacyclin than resting HUVEC. Resting hybrid cells produced more PGE(2) than prostacyclin, despite they expressed high levels of COX-1 and PGI(2) synthase. On the contrary to HUVEC and A549, EA.hy 926 cells did not respond to IL-1beta with COX-2 induction and increase of prostaglandin production, however they did it in response to lysophosphatidylcholine (LPC). The characteristics of EA.hy 926 cells in terms of the pattern of prostanoid formation could facilitate studies on endothelial metabolism and role of these important lipid mediators.     

Regulation of types I and III NOS in ovine uterine arteries by daily and acute estrogen exposure

Nitric oxide contributes to estrogen-mediated uterine vasodilation; however, the nitric oxide synthases (NOS) involved and their location within uterine arteries are incompletely documented. We investigated the effects of repetitive daily and acute estradiol-17beta (E(2)beta) exposure on uterine hemodynamics and NOS abundance and localization in uterine arteries from nonpregnant ovariectomized ewes receiving daily intravenous E(2)beta (1 microg/kg, n = 5) or no E(2)beta (n = 7) for 5 days to determine NOS abundance, cGMP contents, and NOS immunohistochemistry. Daily E(2)beta increased basal and E(2)beta-mediated rises in uterine blood flow (UBF) 36 and 43% (<0.01), respectively, calcium-dependent NOS activity 150% (P < 0.02) in endothelium-intact and -denuded ( approximately 40% of total NOS) arteries, and cGMP contents 39% (P < 0.05). Endothelial (eNOS) was detected in luminal endothelium, whereas neuronal NOS (nNOS) protein was only in the media. A second group of ewes received E(2)beta (1 microg/kg iv) for 4 days and acute intravenous E(2)beta (n = 8) or vehicle (n = 4) on day 5. UBF rose 5.5-fold (P < 0.001) 115 min after E(2)beta, at which time only endothelium-derived calcium-dependent NOS activity increased 30 +/- 13% (P < 0.05). Daily E(2)beta enhances basal and E(2)beta-mediated increases in UBF, which parallel increases in endothelium-derived eNOS and smooth muscle-derived nNOS. Acute E(2)beta, however, selectively increases endothelium-derived eNOS.     

Global protein expression profiling underlines reciprocal regulation of caveolin 1 and endothelial nitric oxide synthase expression in ovariectomized sheep uterine artery by estrogen/progesterone replacement therapy

Ovariectomized (OVX) ewes were assigned to receive vehicle, progesterone (P4, 0.9-g controlled internal drug release vaginal implants), estradiol-17beta (E2, 5 microg/kg bolus + 6 microg kg(-1) day(-1)), or P4 + E2 for 10 days (n = 3/group). Uterine artery endothelial proteins were mechanically isolated on Day 10. The samples were used for protein expression profiling by the Ciphergen Proteinchip system and immunoblotting analysis of endothelial nitric oxide synthase (NOS3, also termed eNOS) and caveolin 1. Uterine artery rings were cut and analyzed by immunohistochemistry to localize NOS3 and caveolin 1 expression. With the use of the IMAC3 protein chip with loading as little as 2 microg protein/sample, many protein peaks could be detected. Compared to vehicle controls, a approximately 133.1-kDa protein was identified to be upregulated by 2- to 4-fold in OVX ewes receiving E2, P4, and their combination, whereas a approximately 22.6-kDa protein was downregulated by 2- to 4-fold in OVX ewes receiving E2 and E2/P4, but not P4 treatments. Western blot analysis revealed that E2, P4, and their combination all increased NOS3 protein, whereas E2 and its combination with P4, but not P4 alone, downregulated caveolin 1 expression. Immunohistochemical analysis revealed that NOS3 was mainly localized in the endothelium and upregulated by E2, whereas caveolin 1 was localized in both endothelium and smooth muscle and downregulated by E2. Thus, our data demonstrate that uterine artery endothelial NOS3 and caveolin 1 are regulated reciprocally by estrogen replacement therapy. In keeping with the facts that E2, but not P4, causes uterine vasodilatation and that E2 and P4 increase NOS3 expression, but only E2 decrease caveolin 1 expression, our current study suggests that both increased NOS3 expression and decreased caveolin 1 expression are needed to facilitate estrogen-induced uterine vasodilatation.     

Interferon-gamma and interleukin 4 inhibit interleukin 1beta-induced delayed prostaglandin E(2)generation through suppression of cyclooxygenase-2 expression in human fibroblasts

Interleukin (IL-)1 stimulates prostaglandin E(2)(PGE(2)) generation in fibroblasts, and preferential couplings between particular phospholipase A(2)(PLA(2)) and cyclooxygenase (COX) isozymes are implicated with IL-1-induced delayed PGE(2)generation. The regulatory effects of interferon (IFN)-gamma and IL-4 on IL-1beta-induced COX, PLA(2)isoforms expression and terminal delayed PGE(2)generation were examined in three types of human fibroblasts. These human fibroblasts constitutively expressed cytosolic PLA(2)(cPLA(2)) and COX-1 enzymes, and exhibited delayed PGE(2)generation in response to IL-1beta. IL-1beta also stimulated expression of cPLA(2)and COX-2 only, while constitutive and IL-1beta-induced type IIA and type V secretory PLA(2)s (sPLA(2)s) expression could not be detected. A COX-2 inhibitor and cPLA(2)inhibitor markedly suppressed the IL-1beta-induced delayed PGE(2)generation, while a type IIA sPLA(2)inhibitor failed to affect it. IFN-gamma and IL-4 dramatically inhibited the IL-1beta-induced delayed PGE(2)generation; these cytokines apparently suppressed IL-1beta-stimulated COX-2 expression and only weakly suppressed cPLA(2)expression in response to IL-1beta. These results indicate that IL-1beta-induced delayed PGE(2)generation in these human fibroblasts mainly depends on de novo induction of COX-2 and cPLA(2), irrespective of the constitutive presence of COX-1, and that IFN-gamma and IL-4 inhibit IL-1beta-induced delayed PGE(2)generation by suppressing, predominantly, COX-2 expression.