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PubMed2000

The characterization of human amnion epithelial and mesenchymal cells: the cellular expression, activity and glucocorticoid regulation of prostaglandin output.

The amnion, a single layer of epithelial cells (EC) overlying layers of mesenchymal cells (MC) has been identified as a source of intrauterine prostaglandins (PG). The objectives of the present study were: (1) to establish a technique for the isolation and culture of pure amnion EC and MC preparations, (2) to characterize the cellular expression of PGHS-II and PGHS activity within these separated amnion cells and (3) to characterize the pattern of glucocorticoid stimulation of these separated amnion cells. Term gestation human amnion was collected after elective caesarean section or vaginal delivery. A trypsin digestion was used to isolate EC and a mechanical digestion and collagenase dispersion was used to isolate MC. Following 48 or 96 h in culture, cells were incubated for 24 h in the presence or absence of 1 microm arachidonic acid and treated with cortisol (F: 10-1000 nm) or 1 microm dexamethasone (DEX). Cell types were identified by immunohistochemistry (IHC). Immunoreactive PGHS-II (ir-PGHS-II) and glucocorticoid receptor (ir-GR) were localized by IHC. PGHS activity was measured as PGE(2)output determined by radioimmunoassay. Mean PGE(2)production by MC at 72 h was 22-fold greater (P<0.05) and at 120 h was 32-fold greater (P<0.03) than PGE(2)output by EC. Administration of arachidonic acid stimulated a 5.0-fold increase in PGE(2)output (P<0.0002) by EC after 72 h and a 3.6-fold increase (P<0.05) after 120 h but did not alter MC PGE(2)output. Despite exogenous substrate, EC PGE(2)output remained significantly less than PGE(2)output by MC. There was no difference in PG production by EC and MC with the onset of labour. Ir-GR expression was found in both EC and MC. F and/or DEX with and without arachidonic acid (AA) stimulated PGE(2)output by EC. Only DEX and not F increased PGE(2)output by MC. These data suggest that relatively pure EC and MC preparations can be established from amnion. PG output and its regulation appears to differ within these two amnion cell types, dependent upon (1) substrate availability and (2) the regulation of PGHS activity.

Adult↗
PubMedDate not supplied

Physiological apoptotic agents have different effects upon human amnion epithelial and mesenchymal cells.

Foetal membrane rupture is thought to follow from gene-controlled tissue remodelling and apoptosis. We reported previously that staurosporine, cycloheximide, actinomycin D, as well as more physiological apoptotic agents (lactosylceramide, 15d-PGJ(2)) increase prostaglandin release in parallel with induction of apoptosis in WISH and amnion epithelial cells. Also, inhibition of prostaglandin release by cyclooxygenase inhibitors or PKA activators is accompanied by a parallel decrease in apoptosis. We hypothesize that amnion prostaglandin metabolism is linked with apoptosis in amnion epithelial cells and thus to membrane rupture. Amnion mesenchymal cells are also critical for membrane integrity. Their susceptibility to apoptotic agents is unknown and is the subject of this report. In amnion epithelial cells, lactosylceramide (125 microM) induced 6.5-fold, 20-fold increases in PGE(2) and NMP production (apoptosis), respectively. Conversely, in mesenchymal cells, lactosylceramide doses up to 200 microM had no effect on PGE(2) or NMP release. In both cell types, incubation with 15d-PGJ(2) (5-100 microM) demonstrated dose and time dependent increases in PGE(2) and NMP. PKA activators inhibited 15d-PGJ(2) induced PGE(2) release and apoptotis in epithelial cells, but not in mesenchymal cells, however. Major amnion cell types have different sensitivities to physiological apoptotic agents. Prostaglandin release occurs coincident with apoptosis in both amnion epithelial and mesenchymal cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Abundant expression of parathyroid hormone-related protein in human amnion and its association with labor.

In animal models, parathyroid hormone-related protein (PTHrP) increases placental calcium transport and inhibits contraction of uterine smooth muscle. The present studies were undertaken to characterize the expression of PTHrP in human uteroplacental tissues. PTHrP mRNA was identified by Northern analysis as a single species (approximately 1.8 kilobases) in human amnion, chorion, placenta, decidua, and myometrium. The most abundant signal was seen in amnion, where it was 10-400 times that in the other uteroplacental tissues. PTHrP mRNA abundance was decreased in amnion (but not in the other tissues) following the onset of labor (P less than 0.001). PTHrP mRNA in amnion appeared to be translated to a bioactive peptide, as PTHrP bioactivity and immunoreactive PTHrP in amnion correlated closely with PTHrP mRNA content (r = 0.86 and 0.95, respectively; P less than 0.05 and P less than 0.01). Amniotic fluid contained PTHrP, 21 +/- 6 pmol/liter (n = 10) at 16 weeks and 41 +/- 9 pmol/liter (n = 7) at 38 weeks (P = 0.05). These concentrations equaled or exceeded those found in plasma of patients with hypercalcemia secondary to PTHrP. After rupture of the fetal membranes, PTHrP mRNA in amnion was decreased by 78% (P less than 0.0001). This decrease appeared to be specific for PTHrP mRNA, as glyceraldehyde-3-phosphate dehydrogenase mRNA was unchanged following rupture of membranes. Like PTHrP mRNA, PTHrP bioactivity and immunoreactive PTHrP in amnion decreased significantly following rupture of membranes (P less than 0.03 and P less than 0.01, respectively). Since PTHrP is a potent antagonist of uterine muscle contraction, the decrease of PTHrP following rupture of the fetal membranes may play a key role in the onset of labor.

Amnion↗
PubMed1983

Phosphorylation of keratin and vimentin polypeptides in normal and transformed mitotic human epithelial amnion cells: behavior of keratin and vimentin filaments during mitosis.

Analysis by means of two-dimensional gel electrophoresis (IEF) of [32P]orthophosphate-labeled proteins from mitotic and interphase transformed amnion cells (AMA) has shown that keratins IEF 31 (Mr = 50,000; Hela protein catalogue number), 36 (Mr = 48,500), 44 (Mr = 44,000), 46 (Mr = 43,500), as well as vimentin (IEF 26; Mr = 54,000) are phosphorylated above their interphase level during mitosis. Similar studies of normal human amnion epithelial cells (AF type) confirmed the above observations except in the case of keratin IEF 44 whose relative proportion was too low to be analyzed. Immunofluorescent staining of methanol/acetone-treated mitotic transformed amnion cells with a mouse polyclonal antibody elicited against human keratin IEF 31 showed a dotted staining (with a fibrillar background) in all of the cells in late anaphase/early telophase (characteristic "domino" pattern) and in a sizeable proportion of the cells in other stages of mitosis. Normal mitotic amnion cells on the other hand showed a fine fibrillar staining of keratins at all stages of mitosis. Similar immunofluorescent staining of normal and transformed mitotic cells with vimentin antibodies revealed a fibrillar distribution of vimentin in both cell types. Taken together the results indicate that the transformed amnion cells may contain a factor(s) that modulates the organization of keratin filaments during mitosis. This putative factor(s), however, is most likely not a protein kinase as transformed amnion cells and amnion keratins are modified to similar extents. It is suggested that in general the preferential phosphorylation of intermediate-sized filament proteins during mitosis may play a role in modulating the various proposed associations of these filaments with organelles and other cellular structures.

Amnion↗
PubMed1977

Amnion nodosum and congenital ichthyosis.

Histologic characteristics of the placentas in four cases of amnion nodosum and congenital ichthyosis, a rare association, are presented. Two cases were missed abortions of a single multiparous woman, supporting the hypothesis that in congenital ichthyosis amnion and skin share an abnormal genetic trait. As the amnion showed no hyperkeratosis, it is likely that the amniotic lesions are related to an increased deposition of keratotic plugs on the amnion. Oligohydramnios was verified in two cases; urinary tract malformations were absent in all. The histologic characteristics of the placentas were similar. Noteworthy was the aspect of the chorionic vessels, whose lumens were reduced or obliterated. Perhaps this feature contributed to the poor nutrition of the amnion, a fact assumed by some authors to explain lesions of amnion nodosum. As these vascular alterations are common after fetal death, it is only in the placenta of the newborn that the vascular changes, compatible with rubella vasculitis, may have altered the nutrition of the amnion.

Amnion↗
PubMed2002

Production of matrix metalloproteinase-9 in lipopolysaccharide-stimulated human amnion occurs through an autocrine and paracrine proinflammatory cytokine-dependent system.

The objective of this study was to determine the presence of autocrine/paracrine regulation of matrix metalloproteinase-9 (MMP-9) expression mediated by proinflammatory cytokines in human fetal membranes. Fetal membranes obtained from women who underwent cesarean delivery before labor were manually separated into amnion and chorion layers and maintained in culture. These explants were stimulated with tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and either lipopolysaccharide (LPS) alone or LPS with anti-TNFalpha or anti-IL-1beta-neutralizing antibodies. Levels of proMMP-9 in culture media were evaluated by zymography. Enzyme-linked immunosorbant assay was performed to measure the quantity of IL-1beta, TNFalpha, and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) after LPS stimulation. ProMMP-9 activity was upregulated after stimulation of the amnion by LPS, TNFalpha, and IL-1beta. The increased activity of proMMP-9 resulting from LPS stimulation in the amnion was blocked by the addition of TNFalpha neutralizing antibody but not with anti-IL-1beta. No significant effect of LPS, TNFalpha, or IL-1beta on proMMP-9 expression was observed in the chorion; however, the chorion produced both cytokines when stimulated with LPS. In contrast, TIMP-1 levels remained unchanged in all cultures incubated in the presence of LPS. Therefore, these data indicate that proMMP-9 is produced by the amnion but not the chorion in response to LPS. Because anti-TNFalpha-neutralizing antibody inhibits proMMP-9 activity in the amnion, TNFalpha appears to upregulate proMMP-9 production by the amnion in an autocrine fashion. Meanwhile, TNFalpha and IL-1beta produced by the chorion may upregulate amnionic proMMP-9 production in a paracrine manner.

Amnion↗
PubMed1997

Interleukin (IL)-6 and IL-8 production by human amnion: regulation by cytokines, growth factors, glucocorticoids, phorbol esters, and bacterial lipopolysaccharide.

Amniotic fluid at term contains high concentrations of interleukin (IL)-6 and IL-8. The source of these cytokines has not been identified, although the fetal membranes (amnion and chorion) are likely contributors. Amnion cytokine production was investigated by using amnion cells isolated by enzymatic digestion (from placentas delivered at term before labor) and cultured in vitro. IL-6 and IL-8 were measured in conditioned media by ELISA. Amnion cells produced detectable amounts of both IL-6 and IL-8 throughout the 7-day culture period. The ratio of IL-8 to IL-6 was approximately 5:1, similar to the ratio found in amniotic fluid. Production of both IL-6 and IL-8 was stimulated in a concentration-dependent fashion by interleukin-1beta (0.1-10 ng/ml), tumor necrosis factor-alpha (1-100 ng/ml), and bacterial lipopolysaccharide (0.1-10 microg/ml), and also by 100 nM phorbol 12-myristate 13-acetate. Epidermal growth factor (1-25 ng/ml) had only minimal effects on amnion cytokine production. Dexamethasone (10 nM) inhibited IL-6/-8 production by approximately 50% throughout the culture period. Production of IL-6/-8 by cultured amniotic fibroblasts, which under basal conditions was much lower than that by epithelial cells, was regulated by all the agents tested in a fashion similar to that of the epithelial cells. These findings suggest that the amnion contributes to the pool of IL-6 and IL-8 in amniotic fluid. We speculate that amnion-derived cytokines might have functions during normal human parturition that are distinct from their conventional roles as inflammatory mediators.

Amnion↗
PubMed2000

Increased axonal regeneration through a biodegradable amnionic tube nerve conduit: effect of local delivery and incorporation of nerve growth factor/hyaluronic acid media.

The authors emphasize the possible pharmacological enhancement of axonal regeneration using a specific growth factor/ extracellular media incorporated in a biodegradable nonneural nerve conduit material. They investigated the early effects on nerve regeneration of continuous local delivery of nerve growth factor (NGF) and the local incorporation of hyaluronic acid (HA) inside a newly manufactured nerve conduit material from fresh human amnionic membrane. Human amnionic membrane contains important biochemical factors that play a major neurotrophic role in the nerve regeneration process. The process of manufacturing a nerve conduit from fresh human amnionic membrane is described. This nerve conduit system was used in rabbits to bridge a 25-mm nerve gap over 3 months. NGF was released locally, over 28 days, at the distal end of the tube via a system of slow release, and HA was incorporated inside the lumen of the tube at the time of surgery. NGF/HA treatment promoted axonal regeneration across the amnionic tube nerve conduit (8,962 +/- 383 myelinated axons) 45% better than the nontreated amnionic tube group (6,180 +/- 353 myelinated axons). The authors demonstrate that NGF/HA media enhances additional axonal regeneration in the amnionic tube nerve conduit. This result is secondary to the effect of the amnion promoting biochemical factors, in combination with the NGF/HA effect on facilitating early events in the nerve regeneration process.

Amnion↗
PubMed1987

Human amnion as a model for IgG transport.

The ability of fresh human amnion to bind and internalize horseradish peroxidase-labeled IgG (IgG-HRP) was examined in an in vitro Ussing chamber system. The amnion demonstrated unique cell membrane receptors for the Fc portion of IgG molecules (Fc gamma R). The Fc gamma R exhibit exquisite specificity and affinity for IgG monomers as demonstrated by staining with labeled IgG. Labeled IgA, IgM, F(ab')2 fragments of IgG, aggregated IgG, and antigen-antibody complexes all failed to bind to the amnionic epithelial cells. Binding was only minimally affected by changes in ionic strength or pH when viewed at the light microscopic level. The Fc gamma R are located on both the apical and basal cell membranes. The binding of IgG-HRP to the amnion cell membrane was detectable within 1 min, and internalization of the ligand occurred within 5 min. No binding of IgG-HRP was observed following treatment of the membrane with 0.25% trypsin for 30 min at room temperature. Incubation of the amnion at 4 degrees C or in the presence of colchicine or cytochalasin D prevented internalization of the IgG-HRP. These experiments demonstrate Fc gamma R on human amnionic epithelial cells that both bind and internalize IgG, thus allowing the amnion to be used as a model system for studying IgG transport.

Amnion↗
PubMed2005

Only the nose knows: penile hemodynamic study of the perineum-saddle interface in men with erectile dysfunction utilizing bicycle saddles and seats with and without nose extensions.

PURPOSE: To investigate the differential impact of straddles (A(ns) and B(ns)) and noseless two-cheek seats (A(ntcs) and B(ntcs)) on penile hemodynamics and perineal compressive forces in subjects who cycle in a stationary bicycle. MATERIALS AND METHODS: Subjects underwent cavernosal artery peak systolic velocity (CAPSV) measurements after intracavernosal injection of vasoactive agents while supine, sitting upright on an examination table, straddling a saddle, sitting on a seat, and again supine. Mean perineal compression pressures recorded while straddling the saddles were compared with those while sitting upright. RESULTS: No differences were found in right and left CAPSV values while supine, sitting upright on an examination table, sitting on a seat, and supine again. Right/left CAPSV (cm/second) values straddling A(ns) and saddle B(ns) (0.7 +/- 2.9/1.5 +/- 6.2 and 0/0, respectively) were significantly lower than values obtained while sitting on A(ntcs) and B(ntcs) (25.6 +/- 13.4/23.8 +/- 12.0 and 17.3 +/- 6.4/18.3 +/- 6.5, respectively) (P < 0.001). Mean perineal compression pressures (mm Hg) on A(ns) and saddle B(ns) (315.2 +/- 39 and 387.9 +/- 64.3, respectively) were significantly higher than values obtained while sitting upright on an examination table (47.6 +/- 5.2 and 46.0 +/- 8.1, respectively) (P < 0.001). CONCLUSIONS: We have identified an objective test to assess if an individual bicycle rider, sitting on a certain shape of bicycle saddle or seat generates sufficient compressive forces at the perineal-saddle interface to obstruct cavernosal arterial inflow. This study also demonstrated that straddling bicycle saddles with nose extensions is associated with perineum-saddle interface compressive pressures that exceed systolic perfusion pressures, significantly diminishing penile hemodynamics.

Adult↗
PubMed1986

An in vitro model for polymorphonuclear-leukocyte-induced injury to an extracellular matrix. Relative contribution of oxidants and elastase to fibronectin release from amnionic membranes.

Alteration of the extracellular matrix by inflammatory cells is believed to be important in both lung injury and the subsequent restoration of lung architecture. Here we describe the results of the interaction between an acellular human amnionic membrane model and stimulated human polymorphonuclear neutrophils (PMN) in vitro. Polymorphonuclear neutrophil suspensions were placed on one surface of the amnion, and either the chemotactic peptide FMLP or the cell membrane activator phorbol myristate acetate (PMA) was placed on the opposite side of the amnion. Stroma and basement membrane sides of the amnion were separately exposed to the PMN. The PMN suspension was removed and centrifuged, and the supernatant was assayed for superoxide anion (O2-.) and for elastase activity. Injury to the acellular amnion was evaluated by transmission electron microscopy and by measurement of fibronectin (FN) released from the membrane matrix. Although both stimulants cause a concentration-dependent release of O2-., only PMA stimulated elastase release. These effects were similar when either the stroma or the basement membrane side was exposed to PMN. PMA-stimulated cells and supernatants from PMA-stimulated cells caused solubilization of membrane at different incubation times. Electron microscopy confirmed the disruption of the basement membrane of the amnion by PMA-stimulated PMN. Oxidant scavengers (SOD and catalase) did not prevent matrix degradation, and elastase inhibition by a specific chloromethylketone inhibitor diminished FN release on both sides of the amnion by activated PMN supernatants, but only on the basement membrane side by intact PMN. We conclude that in this model, elastase rather than oxygen radicals solubilizes FN from the matrix.

Amnion↗
PubMed1995

Prostaglandin endoperoxide-H synthase-1 and -2 messenger ribonucleic acid levels in human amnion with spontaneous labor onset.

Increased prostaglandin (PG) production within the uterine compartment has a pivotal role in the processes leading to labor onset in women. Two PG endoperoxide-H synthase (PGHS) isoenzymes have been identified in a number of cell types. PGHS-1 is constitutively expressed in most cases, whereas PGHS-2 expression is rapidly induced by several agonists. The aims of this study were to determine the levels of PGHS-1 and PGHS-2 expression before and after spontaneous labor (SL) onset in the amnion and to assess the contribution of PGHS-1 and PGHS-2 to enzyme activity. We established and validated ribonuclease protection assays to quantify PGHS-1 and PGHS-2 messenger ribonucleic acid (mRNA) levels in the amnion. PGHS enzyme activity was measured with an established assay. The antisense RNA probes used in the protection assays were generated using human PGHS-1 and PGHS-2 complementary DNAs. These probes specifically detected the 2.8-kilobase mRNA of PGHS-1 and the 4.8-kilobase mRNA of PGHS-2 in amnion RNA samples on Northern blots. We measured mRNA levels in amnion from patients after SL at term and from patients not in labor undergoing elective cesarean section (CS) at term. PGHS-2 mRNA levels were markedly higher after SL compared to levels in CS amnion [5.18 +/- 1.08 (n = 16) and 2.27 +/- 0.50 (n = 15), densitometric units, respectively; P < 0.02], whereas there was no difference in PGHS-1 mRNA levels after labor compared with CS samples. PGHS-2 mRNA levels were also positively correlated with PGHS enzyme activity in 4 separate assays with a total of 25 patients (r = 0.65-0.88; P < 0.05). There was no correlation between PGHS-1 mRNA levels and enzyme activity. We conclude that PGHS-2 mRNA is present in human amnion; its levels are elevated after SL onset, and they are correlated with enzyme activity. The stimulation of PGHS activity at labor onset probably involves increased expression of PGHS-2. The expression of PGHS-1 does not change in association with labor in human amnion.

Amnion↗
PubMed1995

Synthesis of complement proteins in amnion.

The amnion is a metabolically active tissue that has been identified as a site of synthesis of numerous products. We report that amnion tissue explants and amnion-derived epithelial cells synthesize and secrete six proteins of the complement system, C1r, C1s, C1 inhibitor, factor B, C3, and factor H. Synthesis of C2 was minimal and variable, and C5 was not detected. The six synthesized proteins had size and subunit composition characteristic of proteins synthesized in HEp2, a long term cell line derived from malignant epithelial cells. Constitutive and regulated synthesis of five of the six proteins was similar in amnion tissue and cells. However, synthesis of factor B was different in tissue and cells; constitutive synthesis was 12-fold higher in tissue than in cells, and interleukin-1 did not alter synthesis in tissue, but increased synthesis by 11.7-fold in cells. These results indicate that amnion may be a source of complement proteins present in the amnion fluid and may contribute to local host defense along with endometrial glandular epithelial cells, which synthesize C3. Furthermore, our results suggest that amnion tissue is stimulated in vivo to synthesize factor B and cannot respond to interleukin-1 with a further increase in the synthesis rate.

Adult↗
PubMed1997

Lysyl oxidase (ras recision gene) expression in human amnion: ontogeny and cellular localization.

The tensile strength of human fetal membranes is attributable to interstitial collagens of the zona compacta of the avascular amnion. Collagen fiber strength and proteolytic resistance is provided by inter and intramolecular cross-links of collagen fibrils, which are formed in a series of reactions initiated by lysyl oxidase. Lysyl oxidase activity in amnion tissues varied by more than 400-fold in a highly significant inverse manner as a function of gestational age (12-43 weeks). At 12-14 weeks gestation, the levels of lysyl oxidase messenger ribonucleic acid, protein, and activity in amnion are very high. During the second trimester of pregnancy, however, these decline abruptly, and a nadir is reached at about 20-24 weeks gestation, which persists to term. The level of lysyl oxidase messenger ribonucleic acid was greater in amnion mesenchymal cells than in amnion epithelial cells. The decline in lysyl oxidase in amnion may be attributable to a correspondent decline in the density of amnion mesenchymal cells with fetal development.

Amnion↗
PubMed1997

Keratinocyte growth factor expression in the mesenchymal cells of human amnion.

Amnion epithelial and mesenchymal cells were separated by differential protease treatment, and the separated cells were maintained in monolayer culture. Keratinocyte growth factor (KGF) messenger RNA (mRNA) was readily detected by Northern analysis of amnion mesenchymal cell total RNA (10 micrograms) but not in amnion epithelial cells. Treatment of the amnion mesenchymal cells in serum-free medium with tetradecanoyl phorbol acetate (1 nM) caused an increase in the level of KGF mRNA. Forskolin treatment also caused an increase in KGF mRNA but not to the levels attained with tetradecanoyl phorbol acetate treatment. Dexamethasone (1 nM) treatment of these cells effected a reduction in the level of KGF mRNA. Prolonged maintenance of mesenchymal cells in serum-free medium also was associated with an increase in the level of KGF mRNA. Treatment with a variety of other agents, viz., interleukin (IL)-1, IL-6 plus or minus IL-6 soluble receptor, IL-11, oncostatin M, epidermal growth factor (EGF), and transforming growth factor-beta and not modify the level of KGF mRNA. Treatment of amnion epithelial cells with KGF caused an increase in the rate of [3H]thymidine incorporation, but the rate of cell replication induced by KGF was less than that induced by treatment with EGF. Transforming growth factor-beta treatment inhibited basal and EGF- and KGF-stimulated amnion epithelial cell replication. The findings of this study are indicative the KGF is expressed in human amnion mesenchymal cells, and that KGF may act on the epithelial cells of this tissue.

Amnion↗
PubMed1993

Preferential increase in prostaglandin endoperoxide H synthase compared with lipoxygenase activity in sheep placenta and amnion at term pregnancy and after intrafetal glucocorticoid administration.

Prostaglandins (PGs) have been implicated as stimulants to myometrial contractility at parturition in many species. To determine whether the increased production of PGs at parturition reflects a general increase in the metabolism of arachidonic acid or a specific increase in PG endoperoxide H synthase (PGHS) compared with lipoxygenase activities, and to determine intrauterine sites of these activities, we examined the metabolism of [3H]arachidonic acid by homogenates of placenta, amnion and chorion from sheep at days 78-80, 100-105, 135-140 of pregnancy and at term (day 145). Tissues were also obtained from fetuses at day 125; four of these were infused for 84 h with cortisol and four were used as saline-treated controls. The endogenous arachidonic acid content at the start of incubation was measured by capillary gas chromatography. Radioactive metabolites were separated and quantified by reverse-phase high-pressure liquid chromatography. At each gestational age arachidonic acid was converted to PGs, leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs). Conversion to PGs was greater in amnion than in chorion or placenta between days 78 and 140. The formation of PGs rose in placenta at term to a mean value twice that of amnion and ten times that of chorion. In amnion, the ratio of PG:LT rose significantly at term relative to 100-140 days of gestation. In placenta, the ratio of PG:LT produced from arachidonic acid and the ratio of total PGHS:lipoxygenase products rose significantly at term. In the day-125 fetuses treated with cortisol there was a significant increase in PG production relative to that in control fetuses infused with saline in placenta, amnion and chorion; the placenta and amnion being the major sites of PG production. Production of LTs and HETEs also rose significantly in the chorion and the placenta relative to controls. In both the placenta and the amnion there was a significant increase in the ratio of total PGHS to lipoxygenase products formed. We conclude that at term labour and in labour induced by intrafetal cortisol infusion, the placenta is the major site of arachidonic acid metabolism, and that there is a preferential increase in the formation of PGs over lipoxygenase products. These results are consistent with the suggestion that there is an increase in the expression or activity of PGHS in the placenta of sheep in late pregnancy.

Amnion↗
PubMed2003

WISH cells as a model for the "in vitro" study of amnion pathophysiology.

In the course of pregnancy amnion cells produce a number of factors which include cytokines and prostaglandins (PGs) produced in response to autocrine, paracrine and endocrine signals. Recent studies performed by several researchers contributed to elucidate the mechanism through which amnion tissue is involved in the triggering of physiological labor. However, there are other possible functions to be ascribed to amniotic cells, depending on the high number of factors that they produce as well as on the receptors that enable them to act in turn as target. For instance, it has been demonstrated that amnion cells are able to produce lecithin upon the regulation of several factors, such as glucocorticoids and epidermal growth factor, a finding that suggests a protective role of the tissue on fetal pulmonary function. As regards to triggering the uterine contractions, it is accepted that prostaglandin release by amnion cells represents a key event. It is under the control of hormones, growth factors, cytokines and probably PGs themselves. A striking analogy has been found between the mechanism of inflammation and the onset of myometrial activity in labor. In this context, it has been shown that for-Met-Leu-Phe (fMLP), the prototype of a series of formylated peptides traditionally considered chemotactic agents, is also involved in the regulation of amniotic PG release. The similitude between labor and inflammatory response is enforced by the antiprostaglandin action of some classes of antibiotics observed in amnion tissue, that enable them as effective tools against preterm labor, both in the absence and in the presence of infection. As for the mechanisms responsible for the regulation of PG synthesis, some agents act by influencing protein synthesis, while others exert their effects through the production of intracellular second messengers, mainly represented by phosphatidyl-inositol-4-5 bisphosphate and cyclic AMP. The mechanism whereby second messengers induce PG release is not clear, and a crosstalk between the two transduction pathways could be hypothesized. This interaction has extensively been analysed in "WISH" cells, a human amnion-derived cell line, which represent a model for the in vitro study of amnion functions. In the present review, we intend to report the results of the studies regarding the mechanisms through which the control of the above mentioned functions is executed.

Amnion↗
PubMed2006

[Experimental study on porcine keratinocytes cultured and purified rapidly and cocultured on acellular amnion in vitro].

OBJECTIVE: To explore an effective method to culture and purify porcine keratinocytes, to observe the morphological characteristics of porcine keratinocytes growing on acellular amnion and to offer the experimental basis for that the amnion is used for tissue engineering. METHODS: The primary porcine keratinocytes were cultivated with DKSFM (Defined keratinocyte-SFM) containing 10% fetal bovine serum (FBS). The second passage porcine keratinocytes were cultivated with the medium of DKSFM containing different concentrations of FBS. Because of the speciality that keratinocytes stick to flask fast, we purified the keratinocytes by 0.02% EDTA and 0.05% trypsin step by step. The second passage keratinocytes were seeded on amnion, the keratinocytes/amnion composites were observed by dye directly, histopathology and immunohistochemical staining. RESULTS: The proliferation of the primary porcine keratinocytes cultured with the medium of DKSFM containing 10% FBS was fast and the morphological characteristics were good. The cultivated porcine keratinocytes expanded to 60%-70% of the total area of the bottle of the flask after 5 days. The proliferation of the second passage porcine keratinocytes cultivated with the medium that DKSFM containing 5% FBS was faster than the second porcine keratinocytes cultured with the medium of DKSFM containing 10% FBS, or DKSFM without FBS. The proliferation of the second passage porcine keratinocytes cultivated with DKSFM without FBS was the slowest one among the 3 medium. The porcine keratinocytes that were purified by 0.02% EDTA and 0.05% trypsin step by step were got with high pure. After the keratinocytes were cultivated on the surface of amnion 12 days, the keratinocytes form a single layer on the surface of amnion and the cells were polygon and arranged like slab- stone. After 14 and 16 days, the cells contacted more closely. But at 16 days after the cells were seeded, some of the cells got aging. CONCLUSION: To culture primary porcine keratinocytes with the medium that DKSFM containing 10% FBS and to cultivate the second passage with the medium containing 5% FBS, the proliferation of porcine keratinocytes are faster. The method that purify the porcine keratinocytes is effective. Acellular amnion offers excellent bioscaffold to support keratinocytes to adhere and grow. After the porcine keratinocytes are cultivated on the surface of the acellular amnion 12 days, the morphologic characteristics are better than that of other groups.

Actihaemyl↗