Focus: The Science of Stress: Mesenchymal Stem/Stromal Cells and Their Role in Oxidative Stress Associated with Preeclampsia

Preeclampsia (PE) is a serious medically important disorder of human pregnancy, which features de novo pregnancy-induced hypertension and proteinuria. The severe form of PE can progress to eclampsia, a convulsive, life-threatening condition. When placental growth and perfusion are abnormal, the placenta experiences oxidative stress and subsequently secretes abnormal amounts of certain pro-angiogenic factors (eg, PlGF) as well as anti-angiogenic factors (eg, sFlt-1) that enter the maternal circulation. The net effect is damage to the maternal vascular endothelium, which subsequently manifests as the clinical features of PE. Other than delivery of the fetus and placenta, curative treatments for PE have not yet been forthcoming, which reflects the complexity of the clinical syndrome. A major source of reactive oxygen species that contributes to the widespread maternal vascular endothelium damage is the PE-affected decidua. The role of decidua-derived mesenchymal stem/stromal cells (MSC) in normotensive and pathological placenta development is poorly understood. The ability to respond to an environment of oxidative damage is a “universal property” of MSC but the biological mechanisms that MSC employ in response to oxidative stress are compromised in PE. In this review, we discuss how MSC respond to oxidative stress in normotensive and pathological conditions. We also consider the possibility of manipulating the oxidative stress response of abnormal MSC as a therapeutic strategy to treat preeclampsia.     

Megalin in normal tissues and carcinoma cells carries oligo/poly α2,8 deaminoneuraminic acid as a unique posttranslational modification

In rat kidney, megalin, a member of the low density lipoprotein receptor gene family, is the sole glycoprotein which carries oligo/poly 2,8 deaminoneuraminic acid (KDN) as a posttranslational modification. We have investigated immunoprecipitated megalin from rat brain, lung and placenta, mouse yolk sac carcinoma and megalin synthesizing carcinoma cell lines, for presence of this unique glycan structure. Our immunoblot analysis revealed the presence of oligo/poly 2,8 KDN on megalin in all the studied normal tissues and carcinoma cells. Furthermore, it is demonstrated that to be part of oligosaccharides O-glycosidically linked to megalin.     

Venusol from Gunnera perpensa: structural and activity studies

From the aqueous extract of the dry rhizomes of Gunnera perpensa the minor components pyrogallol, succinic acid, lactic acid, and the trimethyl ether of ellagic acid glucoside were isolated. The major constituent was identified as Z-venusol, a phenylpropanoid glucoside. Its structure was verified by X-ray diffraction. Tests on isolated uterine smooth muscle from rats showed that the whole extract stimulated a direct contractile response and induced a state of continuous contractility of the uterus once all additives had been removed from the organ bath. By contrast, venusol did not trigger the direct contractile response but induced the state of continuous contractility once the organ bath was flushed.     

Role of apoptosis in functional luteolysis in the pregnant rabbit corpus luteum: Evidence of a role for placental-derived factors in promoting luteal cell survival

Corpora lutea (CL) were isolated from one rabbit ovary on days 4, 8, 16 (peak luteal function), 28 (functional regression) and 30 of pregnancy and processed for biochemical analysis of DNA integrity. Analysis of DNA integrity revealed the presence of oligonucleosomal fragments in day 28 and day 30 CL but not in day 16 CL. The extent of low molecular weight (<15 kb) DNA labeling was 6.6 +/- 0.84 fold higher in day 30 as compared to day 16 CL (mean +/- SEM; n = 4, P < 0.01). In a second series of experiments, healthy CL collected from day 16 pregnant rabbits were incubated for 2 h in the absence or presence of 250 microg/ml of placental extract (PE) obtained from day 16 and/or day 30 placentas. Analysis of DNA integrity revealed that extensive apoptosis occurred in CL incubated in medium alone and in medium containing day 30 PE. In contrast, day 16 PE significantly suppressed apoptosis vs control (70 +/- 4%). In a third series of experiments, expression of mRNA for bcl-x and bax was measured by Northern analysis of CL treated without and with day 16 PE using cRNA probes for bcl-x and bax developed in our laboratory by RT-PCR. Treatment with PE significantly reduced bax mRNA levels but did not change bcl-x mRNA levels. These studies provide evidence that functional luteolysis in the pregnant rabbit CL is correlated with the occurrence of apoptosis. The data suggest that a factor(s) derived from the placenta may be responsible for the prolongation of CL life span during pregnancy by its ability to alter the bax:bcl -x rheostat and suppress apoptosis.     

Placentation in garter snakes. II. Transmission EM of the chorioallantoic placenta of Thamnophis radix and T. sirtalis

Transmission electron microscopy was used to examine the ultrastructure of the allantoplacenta of garter snakes during the last half of gestation. This placenta occupies the dorsal hemisphere of the egg and is formed through apposition of the chorioallantois to the inner lining of the uterus. The uterine epithelium consists of flattened cells with short, irregular microvilli and others that bear cilia. The lamina propria is vascularized and its capillaries lie at the base of the uterine epithelial cells. The chorionic epithelium consists of a bilayer of squamous cells that are particularly thin superficial to the allantoic capillaries. Neither the chorionic epithelium nor the uterine epithelium undergoes erosion during development. Although a thin remnant of the shell membrane intervenes between fetal and maternal tissue at mid-gestation, it undergoes fragmentation by the end of gestation. Thus, uterine and chorionic epithelial are directly apposed in some regions of the allantoplacenta, forming continuous cellular boundaries at the placental interface. During development, capillaries proliferate in both the uterine and chorioallantoic tissues. By late gestation, the interhemal diffusion distance has thinned in some areas to less than 2 microm through attenuation of the uterine and chorionic epithelia. Morphologically, the allantoplacenta is well adapted for its function in gas exchange. However, the presence of cytoplasmic vesicles, ribosomal ER, and mitochondria in the chorionic and uterine epithelial cells are consistent with the possibility of additional forms of placental exchange.     

Gene for porcine pregnancy-associated glycoprotein 2 (poPAG2): its structural organization and analysis of its promoter

The pregnancy-associated glycoproteins (PAG) are abundant secretory products of the placental trophectoderm of ungulate species. They are structurally related to pepsin, having the capability to bind peptides. However, many cannot function as enzymes due to amino acid substitutions in and around the catalytic site. Here, we demonstrate that pigs, like cattle and sheep, but unlike equids, have multiple PAG genes. One of the transcribed porcine PAG (poPAG) genes, the one for poPAG2, was cloned. It had a nine-exon organization similar to that of other mammalian aspartic proteinase genes with an atypical TATA sequence. A total of 1.2 kbp upstream from exon 1 was sequenced. This region shared identity (> 65%) with the promoter regions of the bovine (bo) PAG1, boPAG2 and equine (eq) PAG genes, but not with other aspartyl proteinase genes, including that of pepsinogen A. Nor were there clear similarities to the promoters of other genes with trophoblast-specific expression. Of the different poPAG2 promoter constructs tested in transfection experiments in two human (JAr and JEG3) and one rat (Rcho) choriocarcinoma cell lines, only the shortest (-149 bp) was required to provide full expression of a luciferase reporter. Although this short promoter was not active in Cos-1 and L-929 cells, it was active in CHO cells, a transformed non-trophoblast hamster ovarian cell line. Co-transfection of Ets2 elevated the activity of this short promoter approximately six-fold in JAr cells, but, disruption of the two putative Ets sites did not alter the ability of Ets2 to transactivate the promoter. In the non-trophoblast cell lines, Ets2 failed to elicit any response. Ets2 responsiveness may be a common feature of most or all trophoblast-expressed genes, although in the case of poPAG2, the effect may be indirect.     

Isolation of endothelial cells from human placental microvessels: effect of different proteolytic enzymes on releasing endothelial cells from villous tissue

Approaches for the isolation of human placental microvascular endothelial cells (HPMEC) using proteolytic enzymes have been described recently. However, the isolation procedure and enzyme composition most suitable for optimal disaggregation of placental tissue and isolation of HPMEC has not yet been established. We tested different proteolytic enzymes and enzyme mixtures for their capabilities of releasing endothelial cells from human term placental villous tissue. Best results were obtained with a mixture of collagenase/dispase/deoxyribonuclease I (0.28%/0.25%/0.01%). By adding a discontinuous Percoll gradient centrifugation step to the enzymatic dispersion, about 1 x 10(6) cells/g tissue with more than 30% von Willebrand factor (vWf)-positive cells were obtained. However, the total cell number and number of vWf-positive cells were highly dependent on the lot of collagenase used. A perfusion step prior to mincing of villous tissue did not increase the amount of vWf-positive cells. We conclude that the methods described in this study are suitable to isolate high yields of HPMEC and that the composition of the collagenase preparation is crucial to the successful release of endothelial cells from placental tissue. To obtain pure HPMEC, further separation steps, e.g., cell sorting with antibodies against endothelial specific cell surface antigens are necessary.     

Subtilisin proprotein convertase-6 expression in the mouse uterus during implantation and artificially induced decidualization

During implantation, a balance of factors regulates the invasive properties of the embryo and the anti-invasive properties of uterine decidua. Although antiproteinases such as the metalloproteinase inhibitor TIMP-3 are thought to play critical roles in preventing the overaggressive invasion of trophoblasts, the mechanism of antiproteinase regulation is unknown. Recently, the prohormone convertase SPC-6 has been found to be co-expressed in embryo-proximal decidua in association with TIMP-3. As members of this serine proteinase family are known to activate latent TGFbeta family members which regulate decidual TIMP-3 levels, we sought to characterize the expression of SPC-6 during pregnancy and artificial decidualization. In this study, we demonstrate that the zone of SPC-6 gene expression exhibits a great degree of temporal and spatial overlap with TIMP-3 gene expression in uterine decidua from E5.5 through to E8.5. Like TIMP-3, we demonstrate that SPC-6 expression is induced during the decidual cell response using an in vivo model of artificial decidualization. Both the secreted and membrane bound forms of SPC-6 are expressed throughout the period of decidualization, suggesting that SPC-6 may play multiple roles during this developmental period. This is confirmed by our observation of the movement of SPC-6 expression to the presumptive placental region, as TIMP-3 expression regresses at the implantation site.     

Expression and localization of angiogenin in placenta: enhanced levels at term over first trimester villi

Human angiogenin, a 14-kDa non-glycosylated polypeptide with both angiogenic and ribonucleolytic activities, is implicated in angiogenesis, a complex process of proliferation and formation of new capillary blood vessels from existing blood vessels. Placental growth requires extensive angiogenesis, which develops its vascular structure in both fetal chorionic villi and maternal deciduas. In this study, we investigated the expression profiles of angiogenin in placental villi from early and late gestation at both mRNA and protein levels using explant cultures in vitro followed by RT-PCR, immunoblot, and immunohistochemical analyses. From functionally active placental explants, angiogenin was detected in conditioned media of all the samples from first trimester and term group. The mean levels of angiogenin produced by term villi were found to be 2.6-, 2.1-, and 2.2-fold higher (P < 0.01) than first trimester villi at 24, 48, and 72 hr of culture, respectively. Expression profiles of angiogenin from term and first trimester villi seem to agree with its mRNA levels and immunoblot analysis; the expression in term villi was twice that in first trimester villi. The presence of angiogenin in placental villi and upregulation of its production towards term indicate that angiogenin production by the placenta is specific to the developmental stage. In conclusion, the observed changes in the localization and mRNA expression of angiogenin during placental development raise the possibility that it is involved in morphological and angiogenic changes in this endocrine organ vital to the successful fetal outcome during pregnancy.