Phylogeography's past, present, and future: 10 years after Avise, 2000.

Approximately 20 years ago, Avise and colleagues proposed the integration of phylogenetics and population genetics for investigating the connection between micro- and macroevolutionary phenomena. The new field was termed phylogeography. Since the naming of the field, the statistical rigor of phylogeography has increased, in large part due to concurrent advances in coalescent theory which enabled model-based parameter estimation and hypothesis testing. The next phase will involve phylogeography increasingly becoming the integrative and comparative multi-taxon endeavor that it was originally conceived to be. This exciting convergence will likely involve combining spatially-explicit multiple taxon coalescent models, genomic studies of natural selection, ecological niche modeling, studies of ecological speciation, community assembly and functional trait evolution. This ambitious synthesis will allow us to determine the causal links between geography, climate change, ecological interactions and the evolution and composition of taxa across whole communities and assemblages. Although such integration presents analytical and computational challenges that will only be intensified by the growth of genomic data in non-model taxa, the rapid development of "likelihood-free" approximate Bayesian methods should permit parameter estimation and hypotheses testing using complex evolutionary demographic models and genomic phylogeographic data. We first review the conceptual beginnings of phylogeography and its accomplishments and then illustrate how it evolved into a statistically rigorous enterprise with the concurrent rise of coalescent theory. Subsequently, we discuss ways in which model-based phylogeography can interface with various subfields to become one of the most integrative fields in all of ecology and evolutionary biology.

Identification and dynamics of a cryptic suture zone in tropical rainforest.

Suture zones, shared regions of secondary contact between long-isolated lineages, are natural laboratories for studying divergence and speciation. For tropical rainforest, the existence of suture zones and their significance for speciation has been controversial. Using comparative phylogeographic evidence, we locate a morphologically cryptic suture zone in the Australian Wet Tropics rainforest. Fourteen out of 18 contacts involve morphologically cryptic phylogeographic lineages, with mtDNA sequence divergences ranging from 2 to 15 per cent. Contact zones are significantly clustered in a suture zone located between two major Quaternary refugia. Within this area, there is a trend for secondary contacts to occur in regions with low environmental suitability relative to both adjacent refugia and, by inference, the parental lineages. The extent and form of reproductive isolation among interacting lineages varies across species, ranging from random admixture to speciation, in one case via reinforcement. Comparative phylogeographic studies, combined with environmental analysis at a fine-scale and across varying climates, can generate new insights into suture zone formation and to diversification processes in species-rich tropical rainforests. As arenas for evolutionary experimentation, suture zones merit special attention for conservation.

Tumour necrosis factor alpha stimulates the production of monocyte chemoattractants by extravillous trophoblast cells via differential activation of MAPK pathways.

The decidual microenvironment is characterized by a unique population of leukocytes composed primarily of CD56(bright) NK cells and macrophages. The latter are situated near trophoblast cells at the fetal-maternal interface and there is evidence that trophoblast cells are capable of recruiting macrophages to this site. This study sought to determine the role of tumour necrosis factor alpha (TNF) in the trophoblast-mediated recruitment of monocyte-derived macrophages to the fetal-maternal interface. The human first trimester extravillous trophoblast cell line HTR-8/SVneo was shown to express TNFR1 and to secrete the monocyte-attracting chemokines CCL2 and CCL5 after exposure to TNF in a dose-dependent manner. TNF-mediated stimulation of CCL2 secretion was completely inhibited by incubating the trophoblast cells with the p38-MAPK inhibitor SB203580, whereas CCL5 secretion was inhibited by treating the trophoblast cells with inhibitors specific for JNK (SP600125) and ERK kinase (U0126). Media conditioned by TNF-treated trophoblast cells significantly enhanced the ability of the monocyte cell line THP-1 to invade through Matrigel, and this effect was inhibited using antibodies specific for CCL2 and CCL5. These results support a role for TNF at the fetal-maternal interface as a regulator of macrophage recruitment by trophoblast cells.

Rapid acquisition of multicellular drug resistance after a single exposure of mammary tumor cells to antitumor alkylating agents.

BACKGROUND: Clinical drug resistance is either intrinsic (de novo) or often acquired rapidly in conjunction with chemotherapy. By contrast, the selection of drug-resistant mutant cell lines in monolayer culture systems is usually a more protracted process. Sublines of mouse EMT-6 mammary tumor cells selected for resistance to various alkylating agents in vivo after serial passage into syngeneic mice manifest their resistance in vitro only when cultured as three-dimensional multicellular aggregates or spheroids. PURPOSE: We examined whether a single exposure of mouse EMT-6 or human MDA-MB-231 breast cancer cells to alkylating agents in vitro is sufficient for the induction of a resistance phenotype, which may be detected by re-applying the drugs to cells grown as three-dimensional aggregates. METHODS: Mouse EMT-6 and human MDA-MB-231 breast cancer cells cultured as three-dimensional aggregates were exposed to a single dose of alkylating agent for 1-5 days. Aggregates were dispersed, and cells were plated as monolayer cultures for up to 8 weeks to allow for recovery. Colony-forming ability was assessed after a subsequent alkylating-agent exposure of cells cultured as monolayers or three-dimensional aggregates. RESULTS: A single in vitro exposure to 12.5-microM cisplatin (CDDP) for 5 days or 25 microM 4-hydroperoxycyclophosphamide (4-O2H CTX) for 1 or 3 days without changing the medium was sufficient to induce transient but substantial resistance in EMT-6 cells as determined by clonogenic assays. Such resistance was not detected when monolayer cell cultures were used. The concentration of 4-O2H-CTX and the length of time the cells remained in three-dimensional culture after initial exposure to this drug was associated with the degree of subsequent drug resistance of cells grown as three-dimensional cultures. Furthermore, this acquired resistance after a single drug exposure was accompanied by changes in the three-dimensional architecture of the cell aggregates, which now formed much more compact multicellular spheroids. Similarly, a single exposure to 4-O2H-CTX was enough to bring about resistance in MDA-MB-231 cells detectable only in three-dimensional cultures, as well as the change in three-dimensional architecture. CONCLUSIONS: Rapid acquisition of resistance likely represents a physiologic mechanism of adaptation operative at the multicellular level rather than a stable genetic change and may be one of the reasons for the rapid development of drug resistance acquired by tumors in vivo. IMPLICATIONS: In vivo drug exposure may result in transient and low levels of drug resistance that may nevertheless be clinically relevant.

Nitric oxide-mediated regulation of chemosensitivity in cancer cells.

BACKGROUND: Hypoxia in tumors is associated with malignant progression, metastatic spread, and increased resistance to radiotherapy and chemotherapy. Molecular O(2) is required for the cellular production of nitric oxide (NO) by the enzyme NO synthase (NOS), and NO may block components of the adaptive response to hypoxia. Hence, we hypothesized that hypoxia increases drug resistance in tumor cells by inhibiting endogenous NO production. METHODS: Human breast carcinoma (MDA-MB-231) and mouse melanoma (B16F10) cells were pre-exposed to 20% O(2), 5% O(2), or 1% O(2), incubated with a pharmacologic inhibitor of endogenous NO production, and then treated with chemotherapeutic agents. Resistance was assessed by colony-formation assays, and western blot analysis was used to measure NOS protein levels. All P values were two-sided. RESULTS: Incubation of MDA-MB-231 tumor cells in 1% O(2) maximally increased their resistance to doxorubicin and 5-fluorouracil by 8.5-fold (P =.002) and 2.3-fold (P =.002), respectively, compared with incubation in 20% O(2). B16F10 mouse melanoma cells preincubated in 1% O(2) (versus 20% O(2)) for 12 hours exhibited a twofold increase in resistance to doxorubicin (P<.001). The rapid acquisition of drug resistance after exposure to 1% O(2) could be mimicked by incubating the MDA-MB-231 cells for 12 hours with the NOS inhibitor N(G)-monomethyl-Larginine (fivefold increase; P<.001). Conversely, replacement of NO activity by use of the NO-mimetic glyceryl trinitrate (GTN) and diethylenetriamine NO adduct produced statistically significant attenuations in the development of resistance of 59% (P<.001) and 40% (P<.001), respectively, in MDA-MB-231 cells. Treatment of B16F10 cells with GTN produced a 58% reduction in resistance (P<.001). MDA-MB-231 cells expressed all three isoforms of the NOS enzyme at levels that were not altered by exposure to hypoxia. CONCLUSIONS: NO mediates chemosensitivity in tumor cells, and hypoxia-induced drug resistance appears to result, in part, from downstream suppression of endogenous NO production. These results raise the possibility that administration of small doses of NO mimetics could be used as an adjuvant in chemotherapy.

Reversal by hyaluronidase of adhesion-dependent multicellular drug resistance in mammary carcinoma cells.

BACKGROUND: De novo or acquired resistance to chemotherapeutic drugs continues to be one of the most important obstacles hindering the successful treatment of cancer patients. Consequently, enhancing the efficacy of conventional chemotherapeutic drugs has become an important research goal. Our previous studies using the mouse EMT-6 mammary carcinoma selected for resistance to various alkylating agents in vivo demonstrated that such acquired drug resistance may be manifested in vitro only in cells growing in a three-dimensional configuration but not in conventional monolayer culture. We also found that this phenomenon, which we refer to as "acquired multicellular resistance," is associated with an increase in intercellular adhesion or compaction of the alkylating agent-resistant cell lines grown as aggregates in three-dimensional culture. PURPOSE: The present study further investigates the impact of three-dimensional architecture on acquired multicellular drug resistance and its influence on cell cycle kinetics, cell cycle arrest, and cell survival. METHODS: To test the hypothesis that an increase in three-dimensional compaction is related to the drug resistance properties of the cells, we did the following: 1) selected clones of the EMT-6 cell line that spontaneously formed tightly or loosely adherent aggregates and assessed their respective drug resistance properties in vitro; 2) assayed tumorigenic potential of the tight and loose clones after exposure to defined concentrations of the activated form of cyclophosphamide, 4-hydroperoxycyclophosphamide (4-HC) in vitro; and 3) treated the tight clones with hyaluronidase, an agent capable of disrupting EMT-6 spheroids, and assayed what effect this treatment had on chemosensitivity. We used fluorescence-activated cell sorter analysis to monitor any potential alterations in cell cycle kinetics. RESULTS: The increase in compaction in three-dimensional culture was sufficient to confer resistance to 4-HC. This increase in intercellular adhesion was also associated with a lower proliferating fraction of tumor cells and with an almost completely diminished ability of the cells to arrest in the G2/M phase of the cell cycle after drug exposure. Furthermore, these changes were detectable only in three-dimensional culture, not in conventional monolayer culture. In conventional monolayer culture, all cell types consistently showed a high level of proliferation and arrested in G2/M after exposure to 4-HC. Moreover, hyaluronidase was able to disrupt intercellular adhesion and chemosensitize tumor cells both in vitro and in vivo in an ascites model. CONCLUSION: Earlier studies have demonstrated that hyaluronidase is able to sensitize tumor cells to various anticancer agents. Our studies now demonstrate that this sensitization can occur by a mechanism independent of increased drug penetration. This mechanism is likely to be related to the "anti-adhesive" effect of hyaluronidase, which overrides cell contact-dependent growth inhibition, recruits cells into the cycling pool, and renders tumor cells more sensitive to cytotoxic agents that preferentially kill rapidly dividing cells. IMPLICATIONS: Other tumor-specific "anti-adhesives" should be explored that can be effective chemosensitizers when used in combination with cell cycle-specific drugs for the treatment of small, solid tumors.

Suppression of invasion by inducible expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in B16-F10 melanoma cells.

BACKGROUND: We have previously shown that down-modulation (i.e., by antisense expression vector) of tissue inhibitor of metalloproteinase-1 (TIMP-1) in a noninvasive, nontumorigenic cell line led to an acquisition of an invasive, tumorigenic, and metastatic ability in these cells. PURPOSE: Our purpose was to examine whether increased levels of murine TIMP-1 can directly suppress the invasive ability of malignant cells. METHODS: Murine B16-F10 melanoma cells were transfected with an expression vector to overproduce TIMP-1. Among these transfectants, we isolated five clonal cell lines (2-5, 2-8, 2-10, 6-5, and 6-9) that showed upregulation (i.e., overexpression) of TIMP-1. RESULTS: These cell lines had an increased basal level of TIMP-1 messenger RNA. TIMP-1 expression was under the control of the mouse metallothionein-I promoter, and four of these five clones (2-5, 2-8, 6-5, and 6-9) showed a threefold to 10-fold induction of TIMP-1 message when they were treated with 20 microM cadmium for 4 hours. An increase in TIMP-1 message led to an increase in TIMP-1 protein activity measured in the conditioned medium of clones 2-10 and 6-5. The invasive ability of the TIMP-1-upregulated cells was tested in a matrigel transwell invasion assay. All of the upregulated clones showed a significant reduction in their invasive ability, relative to the invasive ability of parental B16-F10 and the control 1-2 cell line; this reduction correlated with the level of TIMP-1 overexpression. Cadmium induction of TIMP-1 messenger RNA resulted in a further suppression of the invasive ability of the two inducible cell lines tested (i.e., 2-8 and 6-5). CONCLUSIONS: Our data demonstrate that a specific upregulation of murine TIMP-1 expression in B16-F10 melanoma cells directly suppresses their invasive ability.

Low oxygen tension induces Krüppel-Like Factor 6 expression in trophoblast cells.

The transcription factor Krüppel-Like Factor 6 (KLF6) has important roles in cell differentiation, angiogenesis, apoptosis, and proliferation. Furthermore, there is evidence that KLF6 is required for proper placental development. While oxygen is a critical mediator of trophoblast differentiation and function, the involvement of oxygen in the regulation of KLF6 expression remains unexplored. In the present study we examined the expression of KLF6 in placental tissue from uncomplicated and preeclamptic pregnancies, the latter often characterized by an inadequately perfused placenta. We also determined the effect of hypoxia and the involvement of Hypoxia-Inducible Factor 1α (HIF-1α) on the expression of KLF6 in cultured trophoblast cells and placental tissues. Results revealed that villous, interstitial and endovascular extravillous cytotrophoblasts from placentas from normal and preeclamptic pregnancies express KLF6. In addition, KLF6 immunoreactivity was higher in the placental bed of preeclamptic pregnancies than in those of uncomplicated pregnancies. We demonstrated that hypoxia induced an early and transient increase in KLF6 protein levels in HTR8/SVneo extravillous cytotrophoblast cells and in placental explants. Reoxygenation returned KLF6 protein to basal levels. Moreover, hypoxia-induced up-regulation of KLF6 expression was dependent on HIF-1α as revealed by siRNA knockdown in HTR8/SVneo cells. These results indicate that KLF6 may mediate some of the effects of hypoxia in placental development. The regulation of KLF6 protein levels by oxygen has significant implications for understanding its putative role in diseases affected by tissue hypoxia.

Aberrant maternal inflammation as a cause of pregnancy complications: A potential therapeutic target?

Pre-eclampsia (PE), fetal growth restriction (FGR), pre-term labour and fetal death are common complications of pregnancy often associated with abnormal maternal inflammation. Though the precise causes of these complications remain obscure, altered maternal blood flow to the placenta is an underlying hallmark, especially with respect to the pathogenesis of PE, FGR and fetal demise. Furthermore, deficient trophoblast-mediated spiral artery remodelling is often cited as the primary cause of impaired utero-placental perfusion. Considerably less attention has been directed towards investigating other factors, including maternal vasoconstriction or hemostatic alterations, as contributors to poor utero-placental perfusion. This review provides a rationale for investigating the role of abnormal maternal inflammation in the pathophysiology of pregnancy complications including PE, FGR and fetal demise. In particular, the association between aberrant maternal inflammation and inadequate utero-placental perfusion is considered in the context of inflammation-associated alterations in maternal hemostasis and vasoconstriction. Finally, the role of aberrant maternal inflammation as a cause of local oxidative/nitrosative stress is examined and the possibility of targeting deficient nitric oxide signalling as a therapeutic intervention for the treatment of inflammation-associated pregnancy complications is discussed.

A marked disparity between the expression of prion protein and its message by neurones of the CNS.

Expression of the normal cellular form of prion protein is both necessary and rate-limiting in the spread of prion disease, yet its cellular expression in vivo is poorly understood. To optimise immunohistochemical labelling of this protein in mouse brain, we have developed novel antibodies that recognise cellular prion protein in glutaraldehyde-fixed tissue. Expression was found to be predominantly neuronal, and to differ between different classes of neurone. Thus, neurones immunoreactive for GABA expressed very high levels of normal prion protein; most projection neurones expressed much lower levels, particularly on their axons in the major fibre tracts, and some neurones (e.g. those positive for dopamine) displayed no detectable prion protein. In marked contrast, all neurones, even those that were immunonegative, expressed high levels of message for prion protein, shown by non-radioactive in situ hybridisation. Glia expressed very low levels of message, and undetectable levels of prion protein. We conclude that the steady-state level of prion protein, which differs so markedly between different neuronal types, is primarily controlled post-transcriptionally, possibly by differences in protein trafficking or degradation. These marked differences in the way different neurones produce and/or degrade their normal cellular prion protein may influence the selective spread and neurotoxic targeting of prion diseases within the CNS.

Effect of transforming growth factor-beta on the plasminogen activator system in cultured first trimester human cytotrophoblasts.

Plasminogen activators (PAs) play an important role in facilitating trophoblast invasion of the uterus and in the maintenance of blood fluidity within the placental intervillous spaces. We previously found that transforming growth factor-beta (TGF-beta), produced mainly by the decidua, inhibits first trimester trophoblast invasiveness at least partly via induction of tissue inhibitor of metalloproteinases-1 production and secretion by the trophoblasts. The present study examined whether TGF-beta 1 affects PA and plasminogen activator inhibitor-1 (PAI-1) production by cultured human cytotrophoblasts. Immortalized first trimester human cytotrophoblasts (HTR-8/SVneo) were cultured in the absence or presence of TGF-beta 1 (0-10 ng/ml) for 24 h, after which the levels of urokinase-type PA (uPA), PAI-1 and uPA activity in the serum-free conditioned media were determined by enzyme-linked immunosorbent assay (ELISA), protein zymography, and a chromogenic uPA activity assay. Cellular PAI-1 mRNA levels were also determined in cultures following a 24-h incubation with a single dose (5 ng/ml) of TGF-beta 1. Presence of TGF-beta 1 at 1 ng/ml resulted in a greater than 12-fold reduction in the levels of total uPA as determined by ELISA. Furthermore, released uPA activity levels in similar cultures incubated with 5 ng/ml of TGF-beta 1 were reduced to 35 per cent of control values. In contrast, cultures incubated with as little as 0.1 ng/ml TGF-beta exhibited a 63 per cent increase in the levels of secreted PAI-1 protein. Similarly, both the 2.2- and 3.0-kb PAI-1 cellular mRNA species were elevated in trophoblast cells incubated with 5 ng/ml TGF-beta 1 when compared with control cells. Thus, it appears that the reduced uPA activity observed in the cultures incubated with TGF-beta 1 is due to reduced secretion of uPA and increased PAI-1 production and secretion. These results suggest that TGF-beta may also exert its anti-invasive effect by down-regulating trophoblast-derived PA activity. Through its effects on the PA system, TGF-beta may also play an indirect role in the regulation of uteroplacental blood flow.

Cellular localization of gp46 at the human fetal-maternal interface.

gp46 is a collagen-binding heat-shock glycoprotein with a possible role in the biosynthesis of collagen as well as in cell differentiation and fusion. In this study, the relative levels of gp46 protein and its mRNA transcript were examined, as well as the mRNA levels of collagen types I and IV in first trimester and term human placental tissues. Western blot analysis revealed substantially higher levels of gp46 in first trimester placentae than in term placentae. Similarly, elevated levels of type IV collagen transcript were detected in first trimester relative to term issues. Interestingly, the levels of gp46 and type I collagen mRNA remained unchanged. Immunohistochemical analysis of first trimester tissues demonstrated intense gp46 staining in mononucleated villous and extravillous cytotrophoblasts, decidual cells and in the villous connective tissue stroma. Syncytiotrophoblast in the same tissues also exhibited gp46 staining but at a reduced intensity. In chronic villi of term placentae, faint gp46 staining was only observed in the syncytiotrophoblast layer. However, as in the first trimester placentae, intense labelling was evident in the extravillous cytotrophoblasts and decidual cells of these tissues. These results suggest a developmental regulation of gp46 expression at the fetal-maternal interface during pregnancy and suggest a possible functional link between gp46 and collagen type IV. during gestation.

Does nitric oxide play a role in the aetiology of pre-eclampsia?

Although progress has been made toward our understanding of the pathophysiology of pre-eclampsia, the precise aetiology of this disease still remains an enigma. One of the hallmarks of pre-eclampsia is a failure of the extravillous cytotrophoblast cells to invade and remodel the uterine spiral arterioles during the first trimester of pregnancy. Moreover, studies suggest that the cause of this disorder may be immunological in nature. Evidence is provided here suggesting that impaired trophoblastic invasion of the spiral arterioles may be linked to the altered immunological response associated with pre-eclampsia. Previous studies by Reister et al., 1999 demonstrated a direct relationship between macrophage infiltration of the myometrial segments of spiral arterioles and reduced trophoblastic invasion in pre-eclampsia. Also, it is well established that activated macrophages produce large amounts of nitric oxide (NO). Our present findings reveal that low concentrations of NO-mimetic drugs (glyceryl trinitrate and sodium nitroprusside) inhibit the ability of trophoblast cells to penetrate through reconstituted extracellular matrix (Matrigel). This inhibition is accompanied by a reduced expression of the cell surface urokinase receptor, a molecule important for invasion. These results suggest a possible mechanistic link between the aberrant macrophage infiltration associated with pre-eclampsia, and the maladapted uteroplacental arterioles that characterize the disease.

Differential display analysis of oxygen-mediated changes in gene expression in first trimester human trophoblast cells.

Physiologic or pathologically induced periods of exposure to relatively low levels of oxygen during pregnancy affect the expression and function of certain genes in the placenta. In this study, the differential display technique was utilized to identify genes that are regulated in cultured cytotrophoblast cells by exposure to low levels of oxygen. Using this approach, four genes, which have been designated HRF-1, HRF-2, HRF-6, and HRF-8, were cloned and partially characterized. Northern blot analysis showed that clones HRF-1 and HRF-2 were downregulated in response to exposure to low levels of oxygen, whereas expression of HRF-6 and HRF-8 was increased. DNA sequencing and sequence analysis revealed that HRF-1 may represent an alternatively spliced or tissue-specific form of the Kruppel family zinc finger protein znfp104 gene. Clone HRF-2 showed a high degree of identity with exons 9, 10 and 11 of N33, a gene that is located within a homozygously deleted region of metastatic prostate cancer. Clones HRF-6 and HRF-8 did not exhibit significant sequence identity with known sequences in GenBank and may represent novel genes. None of these genes have previously been shown to be present in trophoblast cells, nor have their expressions been shown to be regulated by oxygen. This study demonstrates that the differential display technique is a novel and effective method to analyse oxygen-mediated changes in gene expression in trophoblast cells.

Adriana and Luisa Castellucci award lecture 1999: role of oxygen in the regulation of trophoblast gene expression and invasion.

Changes in oxygen levels characterize normal and pathological human placentation. For example, relatively low Po(2)values are present around the blastocyst during implantation and in the placenta of the first trimester of pregnancy, a time of maximal trophoblast invasion. Our studies have revealed that low oxygen levels stimulate the in vitro invasiveness of cultured first trimester trophoblasts. This increased invasive ability is linked to elevated expression of some components of the plasminogen activator system and requires the participation of a putative haem protein. As gestation proceeds beyond the first trimester, and the extent of trophoblast invasion decreases, placental oxygen levels rise with a corresponding increase in blood flow. However, during certain pathological conditions, such as pre-eclampsia/intrauterine growth restriction, impaired remodelling of the uterine spiral arterioles leads to vessels with reduced diameters and localized regions of placental ischaemia/hypoxia. Placental hypoxia in the second half of gestation, as a consequence of reduced uteroplacental blood flow, may result in aberrant expression of genes that contribute to the pathophysiology of pre-eclampsia. Some of these genes encode certain cytokines and vasoactive molecules. We have also identified other genes whose expression is regulated by oxygen. Expression of one of them is induced in trophoblast and other cell types cultured under low oxygen levels and the product of the gene is a 43-kDa protein which we have termed PROXY-1. Compared to placental tissues and membranes isolated from uncomplicated pregnancies, PROXY-1 expression is elevated in tissues from pre-eclamptic pregnancies such as chorionic villi of peri-infarct regions, basal plate and membrane decidua, as well as chorion. Overall, these observations suggest that oxygen levels play an important role in placentation and in the pathophysiology of certain complications of pregnancy.

Carbon monoxide decreases perfusion pressure in isolated human placenta.

Carbon monoxide (CO) is one of the metabolites formed via heme oxidation catalysed by the enzyme heme oxygenase (HO). Endogenous formation of CO, mediated by HO, has been noted in both placental and umbilical vessels. In blood vessels from different mammalian sources, it has been proposed that the vasodilator effect of CO is mediated via stimulation of soluble guanylyl cyclase (sGC) and consequent increased cGMP formation. The purpose of the present study was to determine the effect of exogenous CO on placental cotyledon perfusion pressure and to determine the role of sGC in the CO-induced decrease of perfusion pressure using the in vitro human placental perfusion preparation. A thromboxane A2 mimetic (U46619) was added to the foetal perfusion medium to constrict the placental blood vessels. Carbon monoxide was added to the foetal perfusion medium in increasing concentrations to determine its effect on placental perfusion pressure. Carbon monoxide produced a concentration-dependent decrease in placental perfusion pressure. The addition of ODQ, a sGC inhibitor, attenuated the CO-induced decrease in placental perfusion pressure, while addition of YC-1, an activator of sGC, augmented the CO-induced decrease in placental perfusion pressure. The data indicate that CO causes vasorelaxation of placental resistance blood vessels, in large part, via activation of sGC.

Reducing agent and tunicamycin-responsive protein (RTP) mRNA expression in the placentae of normal and pre-eclamptic women.

Recently, the gene encoding a new stress-induced protein termed reducing agent and tunicamycin-responsive protein (RTP) was identified. The function of RTP is unknown, however, the strong upregulation of RTP during cellular differentiation, and exposure to stress conditions including hypoxia suggests a specific role for RTP in these processes. In pre-eclampsia, impaired spiral artery remodelling and reduced perfusion may reduce oxygen tension in the placenta and thereby alter trophoblast differentiation and function. We therefore hypothesized that the expression of RTP mRNA is altered in the placentae of women with pre-eclampsia. The aims of this study were to determine the regional distribution and cellular localization of RTP mRNA expression and compare mRNA abundance in different regions of normotensive control and pre-eclamptic placentae. In normal and pre-eclamptic placentae, RTP mRNA was expressed in the syncytiotrophoblasts and in the intermediate trophoblasts of the basal plate. In early onset pre-eclampsia, RTP mRNA was more abundant in the chorionic villi regions. A further increase was localized to the syncytial knots and to the trophoblasts in the peri-infarct regions. The increased RTP expression may reflect lower oxygen tension and/or other stress stimuli in the placenta in pre-eclampsia.

Characteristics of trophoblast cells migrating from first trimester chorionic villus explants and propagated in culture.

We developed a method of propagating pure first trimester human trophoblast cells growing out of primary explants of mechanically derived chorionic villus fragments (Yagel et al, 1989; Graham et al, 1992). We have now extensively characterized these cells during their initial outgrowth and in long-term culture, employing a variety of markers and techniques as outlined below. By double label immunofluorescence using epithelial (cytokeratin) and mesenchymal (vimentin) cell markers, we identified the chorionic villus migrant cell populations as pure trophoblast (39 per cent of outgrowths) or a mixture of trophoblast and fibroblast (61 per cent). Further phenotyping of the pure trophoblast outgrowths by double label immunostaining using anti-cytokeratin antibody and a panel of other primary antisera revealed that these cells exhibit a variety of markers characteristic of extravillous invasive trophoblast cells in situ: insulin-like growth factor (IGF)-II, NDOG-5, proliferating cell nuclear antigen (PCNA), human leucocyte antigen framework antigen (W6/32) and a distinct set of integrins including alpha 1, alpha 3, alpha 5, alpha v and beta 1 subunits and alpha v beta 3/beta 5 vitonectin receptor. They were negative for alpha 6 and beta 4 integrin subunits. Immunogold electron microscopy of explants grown on type IV collagen gel revealed the production of conventional and oncofetal types of fibronectin by mononucleate trophoblast cells and human placental lactogen by multinucleate cells. Immunolabelling, flow cytometry and immunoprecipitation revealed that this phenotypic profile was retained with complete fidelity in the long-term culture; thus, trophoblasts migrating out of first trimester chorionic villus explants and their propagated progeny belong to the invasive extravillous trophoblast of the placenta.

Heme oxygenase and nitric oxide synthase in the placenta of the guinea-pig during gestation.

Nitric oxide (NO) and carbon monoxide (CO) are novel gaseous chemical messengers that play key roles in cell function and cell-cell communication in many organ systems, including the cardiovascular system. Although the presence of NO synthase (NOS) in the placenta and its role in the regulation of fetoplacental and uteroplacental blood flow are well established, little is known about placental expression and activity of heme oxygenase (HO), the enzyme that catalyses the oxidation of heme to CO, biliverdin and iron, during gestation. The objectives of this study were to elucidate the localization of HO-1 and HO-2 isoforms relative to NOS III protein, and to determine the enzymatic activity of HO in the placenta of the guinea-pig during gestation. Placentae were obtained from pregnant guinea-pigs at gestational day (GD) 34, 50, 62 and full term (term, about GD 68). Immunohistochemical localization of HO-1, HO-2 and NOS III protein was conducted using selective polyclonal antibodies. HO activity was determined by using a gas chromatographic method to measure the rate of formation of CO from heme. Faint staining for HO-1 was observed in the adventitial layer of larger fetal blood vessels of the placenta at GD 34. The intensity of this staining was higher at GD 50 and GD 62, and decreased at full term. Similar areas in serial sections of placentae obtained at these selected times during gestation exhibited lower staining intensity when incubated with anti-HO-2 antiserum. Placental HO activity was significantly increased (P<0.05) at GD 62 compared with GD 34, GD 50 and full term. NOS III (endothelial constitutive NOS) staining was highest at GD 34, decreasing thereafter, and was localized mostly to trophoblast lining maternal channels. The data demonstrate that, in the guinea-pig, placental HO and NOS differ in tissue localization during the second half of gestation, with expression of HO protein and its catalytic activity being higher during near-term pregnancy. In a preliminary immunohistochemical investigation of the full-term human placenta, HO-1 protein was localized primarily in the adventitial region of fetal blood vessels of stem chorionic villi. In view of the vasodilator action of CO and NO, the HO and NOS systems may play key roles in the regulation of placental haemodynamics.

Heme oxygenase activity in term human placenta.

Carbon monoxide (CO) is a novel gaseous chemical messenger, formed during heme oxygenase (HO)-catalysed oxidation of heme. CO is proposed to play a key role(s) in cell function in many organ systems, including vasodilator action in the cardiovascular system. Recently, it has been demonstrated that there is expression of HO protein in the human placenta and this appears to have a regulatory role in placental perfusion. The objective of the present study was to determine HO enzymatic activity in vitro in five different regions of term human placenta. HO activity was determined in the microsomal fraction of tissue homogenate by measuring the rate of formation of CO from heme, using a gas-chromatographic method. HO activity, expressed as nmol CO formed/g tissue wet weight/h, was higher (P< 0.05) in the chorionic plate, chorionic villi, basal plate and chorio-decidua compared with the amnion. The finding that HO enzymatic activity is present in different regions of term human placenta supports the concept that the heme-CO (HO) pathway plays a complementary role with the L -arginine-nitric oxide (nitric oxide synthase) pathway in the regulation of placental haemodynamics.