Denise G. Hemmings

Effects of maternal hypoxia or nutrient restriction during pregnancy on endothelial function in adult male rat offspring

Compromised fetal growth impairs vascular function; however, it is unclear whether chronic hypoxia in utero affects adult endothelial function. We hypothesized that maternal hypoxia (H, 12% O2, n= 9) or nutrient restriction (NR, 40% of control, n= 7) imposed from day 15–21 pregnancy in rats would impair endothelial function in adult male offspring (relative to control, C, n= 10). Using a wire myograph, endothelium‐dependent relaxation in response to methacholine was assessed in small mesenteric arteries from 4‐ and 7‐month‐old (mo) male offspring. Nitric oxide (NO) mediation of endothelium‐dependent relaxation was evaluated using Nω‐nitro‐l‐arginine methyl ester (l‐NAME; NO synthase inhibitor). Observed differences in the NO pathway at 7 months were investigated using exogenous superoxide dismutase (SOD) to reduce NO scavenging, and sodium nitroprusside (SNP; NO donor) to assess smooth muscle sensitivity to NO. Sensitivity to methacholine‐induced endothelium‐dependent relaxation was reduced in H offspring at 4 months (P < 0.05), but was not different among groups at 7 months. l‐NAME reduced methacholine sensitivity in C (P < 0.01), H (P < 0.01) and NR (P < 0.05) offspring at 4 months, but at 7 months l‐NAME reduced sensitivity in C (P < 0.05), tended to in NR (P= 0.055) but had no effect in H offspring. SOD did not alter sensitivity to methacholine in C, but increased sensitivity in H offspring (P < 0.01). SNP responses did not differ among groups. In summary, prenatal hypoxia, but not nutrient restriction impaired endothelium‐dependent relaxation at 4 months, and reduced NO mediation of endothelial function at 7 months, in part through reduced NO bio‐availability. Distinct effects following reduced maternal oxygen versus nutrition suggest that decreased oxygen supply during fetal life may specifically impact adult vascular function.

Cohort Profile: The Maternal-Infant Research on Environmental Chemicals Research Platform

BACKGROUND The Maternal-Infant Research on Environmental Chemicals (MIREC) Study was established to obtain Canadian biomonitoring data for pregnant women and their infants, and to examine potential adverse health effects of prenatal exposure to priority environmental chemicals on pregnancy and infant health. METHODS Women were recruited during the first trimester from 10 sites across Canada and were followed through delivery. Questionnaires were administered during pregnancy and post-delivery to collect information on demographics, occupation, life style, medical history, environmental exposures and diet. Information on the pregnancy and the infant was abstracted from medical charts. Maternal blood, urine, hair and breast milk, as well as cord blood and infant meconium, were collected and analysed for an extensive list of environmental biomarkers and nutrients. Additional biospecimens were stored in the studys Biobank. The MIREC Research Platform encompasses the main cohort study, the Biobank and follow-up studies. RESULTS Of the 8716 women approached at early prenatal clinics, 5108 were eligible and 2001 agreed to participate (39%). MIREC participants tended to smoke less (5.9% vs. 10.5%), be older (mean 32.2 vs. 29.4 years) and have a higher education (62.3% vs. 35.1% with a university degree) than women giving birth in Canada. CONCLUSIONS The MIREC Study, while smaller in number of participants than several of the international cohort studies, has one of the most comprehensive datasets on prenatal exposure to multiple environmental chemicals. The biomonitoring data and biological specimen bank will make this research platform a significant resource for examining potential adverse health effects of prenatal exposure to environmental chemicals.

Human Cytomegalovirus-Caused Damage to Placental Trophoblasts Mediated by Immediate-Early Gene-Induced Tumor Necrosis Factor-α

Infection of the fetal epithelium (trophoblast) lining the villous placenta by human cytomegalovirus (HCMV) accompanies placental inflammations and fetal intrauterine growth restriction. However, the consequences of infection on the villous trophoblast have not been explored. We show that HCMV infection of primary immature (cytotrophoblast-like) or mature (syncytiotrophoblast-like) cultures results in loss of half of the cells within 24 hours of virus challenge. Two-color immunofluorescence of HCMV immediate early (IE) gene expression and apoptosis (terminal dUTP nick-end labeling) revealed apoptosis only in uninfected cells. Antibody to tumor necrosis factor (TNF)-alpha completely inhibited infection-induced trophoblast apoptosis and cell loss, as did co-incubation with epidermal growth factor, known to inhibit trophoblast apoptosis. Transfection with HCMV immediate early- (IE)1-72 and IE2-86, but not IE2-55, expression plasmids induced paracrine trophoblast apoptosis inhibitable by epidermal growth factor or antibody to TNF-alpha. These results show that HCMV infection of villous trophoblasts leads to rapid loss of neighboring cells mediated by viral IE protein-induced TNF-alpha secretion. We propose that HCMV infection damages the placental trophoblast barrier by accelerating trophoblast turnover and decreasing its capacity for renewal.

Signal transduction underlying the vascular effects of sphingosine 1-phosphate and sphingosylphosphorylcholine

Two related lysosphingolipids, sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC) mediate diverse cellular responses through signals transduced by either activation of G-protein coupled receptors or possibly by acting intracellularly. Vascular responses to S1P and SPC measured both in vivo and in dissected vessels show predominantly vasoconstriction with some evidence for vasodilation. Although stimulation with S1P or SPC generally leads to similar vascular responses, the signalling pathways stimulated to produce these responses are often distinct. Nevertheless, mobilization of Ca2+ from intracellular stores and influx of extracellular Ca2+, which both increase [Ca2+]i, occur in response to S1P and SPC. Both mobilization of Ca2+ from intracellular stores and influx of extracellular Ca2+ occur in response to S1P and SPC. As well, both S1P and SPC induce Ca2+-sensitization in vascular smooth muscle which is mediated through Rho kinase activation. In the endothelium, S1P and SPC stimulate the production of the vasodilator, nitric oxide through activation of endothelial nitric oxide synthase. This activation occurs through phosphorylation by Akt and through binding of Ca2+-calmodulin upon increased [Ca2+]i. These lysosphingolipids also activate cyclooxygenase-2 which produces prostaglandins with both vasoconstrictor and vasodilator properties. A balance between the signals inducing vasodilation versus the signals inducing vasoconstriction will determine the vascular outcome. Thus, perturbations in S1P and SPC concentrations, relative expression of receptors or downstream signalling pathways may provide a mechanism for pathophysiological conditions such as hypertension. Given this background, recent studies examining a potential role for S1P and SPC in hypertension and vascular dysfunction in aging are discussed.

Sphingosine-1-Phosphate Acts via Rho-Associated Kinase and Nitric Oxide to Regulate Human Placental Vascular Tone

Abstract Sphingosine-1-phosphate (S1P), a bioactive lipid released from activated platelets, has been demonstrated in animal models to regulate vascular tone through receptor-mediated activation of Rho-associated kinase 1 and nitric oxide synthase 3. The role of S1P in regulation of human vascular tone (particularly during pregnancy, with its unique vascular adaptations and localized platelet activation) is unknown. We hypothesized that S1P would constrict small placental arteries through activation of Rho-associated kinases with modulation by nitric oxide. Reverse transcription-polymerase chain reaction of chorionic plate artery preparations detected mRNAs encoding all five receptors for S1P, and S1P induced dose-dependent vasoconstriction of both chorionic plate and stem villous isobarically mounted arteries, which at 10 μmol/L was 32.9% ± 3.86% (mean ± SEM) and 34.6% ± 7.01%, respectively. In stem villous arteries, S1P-induced vasoconstriction was enhanced significantly following inhibition of nitric oxide synthases with NG-nitro-L-arginine methyl ester (100 μmol/L, 52.6% ± 6.28%, P < 0.05). The S1P-induced vasoconstriction was reversed by Y27632, an inhibitor of Rho-associated kinases (10 μmol/L) in both chorionic plate (to 14.9% ± 4.95%) and stem villous arteries (to 2.71% ± 6.13%). The S1P added to alpha-toxin-permeabilized, isometrically mounted chorionic plate arteries bathed in submaximal Ca2+-activating solution induced Ca2+-sensitization of constriction, which was 47.7% ± 10.0% of that occurring to maximal Ca2+-activating solution. This was reduced by Y27632 to 18.4% ± 18.4%. Interestingly, S1P-induced vasoconstriction occurred in all isobarically mounted arteries but was inconsistent in isometrically mounted chorionic plate arteries. In summary, S1P-induced vasoconstriction in human placental arteries is mediated by increased Ca2+-sensitization through activation of Rho-associated kinases, and this vasoconstriction also is modulated by nitric oxide. Identification of these actions of S1P in the placental vasculature is important for understanding both normal and potentially abnormal vascular adaptations with pregnancy.

Polarized Release of Human Cytomegalovirus from Placental Trophoblasts

ABSTRACT Human cytomegalovirus (HCMV) is a ubiquitous infectious pathogen that, when transmitted to the fetus in utero, can result in numerous sequelae, including late-onset sensorineural damage. The villous trophoblast, the cellular barrier between maternal blood and fetal tissue in the human placenta, is infected by HCMV in vivo. Primary trophoblasts cultured on impermeable surfaces can be infected by HCMV, but release of progeny virus is delayed and minimal. It is not known whether these epithelial cells when fully polarized can release HCMV and, if so, if release is from the basal membrane surface toward the fetus. We therefore ask whether, and in which direction, progeny virus release occurs from HCMV-infected trophoblasts cultured on semipermeable (3.0-μm-pore-size) membranes that allow functional polarization. We show that infectious HCMV readily diffuses across cell-free 3.0-μm-pore-size membranes and that apical infection of confluent and multilayered trophoblasts cultured on these membranes reaches cells at the membrane surface. Using two different infection and culture protocols, we found that up to 20% of progeny virus is released but that <1% of released virus is detected in the basal culture chamber. These results suggest that very little, if any, HCMV is released from an infected villous trophoblast into the villous stroma where the virus could ultimately infect the fetus.

Increased Myogenic Responses in Uterine but not Mesenteric Arteries from Pregnant Offspring of Diet-Restricted Rat Dams

Abstract Results of epidemiological and animal studies suggest a link between poor in utero growth and cardiovascular disease in adult offspring. Few studies, however, have examined the effects of maternal undernutrition on the vasculature of pregnant female offspring, and to our knowledge, no studies have examined myogenic responses, which are essential to vascular tone development, in these animal models. Thus, myogenic responses were assessed in radial uterine arteries of pregnant female offspring to determine if diet restriction during pregnancy could contribute to transgenerational effects. These results were compared to those in mesenteric arteries, which greatly contribute to peripheral vascular resistance. Myogenic responses in the presence and absence of inhibitors for nitric oxide synthase (NOS) and prostaglandin H synthase (PGHS) were measured in arteries isolated from pregnant, 3-mo-old female offspring of control-fed (Coff) and globally diet-restricted (DRoff) rat dams. Although no differences were found in pregnancy weight gain, litter size, or fetal weights, placental size was significantly reduced in DRoff compared to Coff. Enhanced myogenic reactivity was observed at the highest pressure tested (110 mm Hg) in uterine, but not in mesenteric, arteries from DRoff compared to Coff. Inhibition of NOS, but not of PGHS, significantly increased myogenic responses in uterine arteries at pressures greater than 80 mm Hg in Coff but, interestingly, not in DRoff compared to untreated uterine arteries. Thus, impaired uterine vascular function in diet-restricted pregnant rat dams, which leads to similar impairment in their pregnant offspring, may be a mechanism through which transgenerational effects of unhealthy pregnancies occur.

Tumor-induced inflammation in mammary adipose tissue stimulates a vicious cycle of autotaxin expression and breast cancer progression

Compared to normal tissues, many cancer cells overexpress autotaxin (ATX). This secreted enzyme produces extracellular lysophosphatidate, which signals through 6 GPCRs to drive cancer progression. Our previous work showed that ATX inhibition decreases 4T1 breast tumor growth in BALB/c mice by 60% for about 11 d. However, 4T1 cells do not produce significant ATX. Instead, the ATX is produced by adjacent mammary adipose tissue. We investigated the molecular basis of this interaction in human and mouse breast tumors. Inflammatory mediators secreted by breast cancer cells increased ATX production in adipose tissue. The increased lysophosphatidate signaling further increased inflammatory mediator production in adipose tissue and tumors. Blocking ATX activity in mice bearing 4T1 tumors with 10 mg/kg/d ONO‐8430506 (a competitive ATX inhibitor, IC90 = 100 nM; Ono Pharma Co., Ltd., Osaka, Japan) broke this vicious inflammatory cycle by decreasing 20 inflammatory mediators by 1.5‐8‐fold in cancer‐inflamed adipose tissue. There was no significant decrease in inflammatory mediator levels in fat pads that did not bear tumors. ONO‐8430506 also decreased plasma TNF‐α and G‐CSF cytokine levels by >70% and leukocyte infiltration in breast tumors and adjacent adipose tissue by >50%. Hence, blocking tumor‐driven inflammation by ATX inhibition is effective in decreasing tumor growth in breast cancers where the cancer cells express negligible ATX.—Benesch, M. G. K., Tang, X., Dewald, J., Dong, W.‐F., Mackey, J. R., Hemmings, D. G., McMullen, T. P. W., Brindley, D. N. Tumor‐induced inflammation in mammary adipose tissue stimulates a vicious cycle of autotaxin expression and breast cancer progression. FASEB J. 29, 3990‐4000 (2015). www.fasebj.org

IFPA Meeting 2008 Workshops Report

Workshops are an important part of the IFPA annual meeting. At the IFPA meeting 2008 diverse topics were discussed in 12 themed workshops. Topics covered included: immunology of placentation; galectins and trophoblast invasion; signaling in implantation and invasion; markers to identify trophoblast subpopulations; placental pathology; placental toxicology; stereology; placental transport of fatty acids; placental mesenchymal stem cells; comparative placentation; trophoblast and neoplasia; trophoblast differentiation. This report is a summary of the various topics covered.

Review: Novel insights into the regulation of vascular tone by sphingosine 1-phosphate

Endothelial dysfunction leading to increased vascular tone is implicated in the pathogenesis of cardiovascular disease, hypertension and pregnancy-related complications like preeclampsia and intrauterine growth restriction. Vascular tone is regulated by a balance between vasoconstrictor and vasodilator signals. Some vascular mediators circulate in blood, whereas others are produced by the endothelium and are delivered to the underlying vascular smooth muscle cells (VSMCs). It is proposed that increased permeability of resistance arteries in preeclampsia allows access of circulating vasoactive factors to VSMCs leading to increased vascular tone. This review focuses on the role of sphingosine 1-phosphate (S1P). This sphingolipid enhances the endothelial barrier, but it can also disrupt the barrier under certain conditions. These S1P-mediated effects on the endothelial barrier have been demonstrated in cultured endothelial cells and in isolated venules. They depend on S1P concentrations, the S1P receptors expressed and the vascular bed. However, no studies have examined if vascular tone is regulated by S1P in resistance arteries through changes in endothelial permeability and the leakage of circulating vasoconstrictors. Our recent studies using the pressure myograph system show that access of infused vasoconstrictors to VSMCs is blocked under low S1P concentrations. Pathophysiological levels of infused S1P disrupt the barrier and maximally increase vascular tone by facilitating access of itself and a co-infused vasoconstrictor to the VSMCs. Interestingly, infusion of an intermediate physiological concentration of S1P showed a small increase in endothelial permeability with controlled leakage of a co-infused vasoconstrictor that led to sub-maximal vascular tone development. These and other studies delineate the important role of S1P in the regulation of vascular tone and emphasize how dysfunction of this regulation can lead to pregnancy-related disorders.