Biomedical subjects

Yoel Sadovsky

Publications and source records attributed to Yoel Sadovsky.

At least 19 recordsLinked to original sources

PubMed2005

Peroxisome proliferator-activated receptor-gamma and retinoid X receptor signaling regulate fatty acid uptake by primary human placental trophoblasts.

CONTEXT: Transplacental transfer of fatty acids from the maternal to the fetal circulation is essential for fetal development. The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) regulates fatty acid transport and storage in adipocytes and other cell types. OBJECTIVE: This study tested the hypothesis that PPARgamma and its heterodimeric nuclear receptor partner, retinoid X receptor (RXR), regulate fatty acid uptake by human trophoblasts. DESIGN: Prospective basic laboratory in vitro research was conducted using primary term human trophoblasts. SETTING: The study was performed in the perinatal biology laboratory of an academic medical center. PATIENTS OR OTHER PARTICIPANTS: Study materials were obtained from healthy pregnant women at a gestational age of 37-41 wk. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASURES: Fat uptake and accumulation in human placental trophoblasts were measured. RESULTS: We initially demonstrated that activation of PPARgamma and/or RXR with selective agonists increased the accumulation of neutral lipids in trophoblasts as well as uptake of free fatty acids. Furthermore, activation of PPARgamma and RXR enhanced the expression of the fat droplet-associated protein adipophilin along with fatty acid transport protein (FATP)4, whereas expression of FATP2 was decreased by activation of RXR. Finally, we found that inhibition of p38 MAPK, which diminishes the activity of PPARgamma in trophoblasts, inhibited fatty acid uptake and blocked the PPARgamma- and RXR-dependent increases in adipophilin and FATP4 expression, yet stimulated the expression of FATP1, FATP2, and FATP3. CONCLUSIONS: These data support a role for PPARgamma and RXR in regulation of fatty acid transport and storage in human placental trophoblasts.

Cells, Cultured↗

PubMed2004

Increased measurement accuracy for sequence-verified microarray probes.

Microarrays have been extensively used to investigate genome-wide expression patterns. Although this technology has been tremendously successful, it has suffered from suboptimal individual measurement precision. Significant improvements in this respect have been recently made. In an effort to further explore the underlying variability, we have attempted to globally assess the accuracy of individual probe sequences used to query gene expression. For mammalian Affymetrix microarrays, we identify an unexpectedly large number of probes (greater than 19% of the probes on each platform) that do not correspond to their appropriate mRNA reference sequence (RefSeq). Compared with data derived from inaccurate probes, we find that data derived from sequence-verified probes show 1) increased precision in technical replicates, 2) increased accuracy translating data from one generation microarray to another, 3) increased accuracy translating data from oligonucleotide to cDNA microarrays, and 4) improved capture of biological information in human clinical specimens. The logical conclusion of this work is that probes containing the most reliable sequence information provide the most accurate results. Our data reveal that the identification and removal of inaccurate probes can significantly improve this technology.

Breast↗

PubMed2004

Homocysteine thiolactone induces apoptosis in cultured human trophoblasts: a mechanism for homocysteine-mediated placental dysfunction?

OBJECTIVE: Hyperhomocystinemia is a thrombophilic condition associated with placental dysfunction. We tested the hypothesis that homocysteine-thiolactone, a metabolite of homocysteine, induces apoptosis in cultured trophoblasts. STUDY DESIGN: Cytotrophoblasts from term human placentas were cultured for 72 hours or less in the presence or absence of 50 to 400 micromol/L homocysteine-thiolactone or 400 micromol/L cysteine (control), with or without vitamin C, vitamin E, folate, or N-acetylcysteine. Cell death was assessed by cellular adenosine triphosphate concentration, medium lactate dehydrogenase level, and immunocytochemical staining for the cleavage products of cytokeratin 18 and poly(adenosine diphosphate ribose) polymerase. Changes in expression of p53, Bcl-2, Bax, and Bak were quantified by Western immunoblotting. RESULTS: Homocysteine-thiolactone induced a concentration dependent increase in total cell death and death by apoptosis, compared with control. Vitamin C ameliorated apoptosis in cytotrophoblasts, whereas N-acetylcysteine mitigated cell death in syncytiotrophoblasts. Apoptosis in both phenotypes occurred with increased expression of p53 and Bak, but no change in Bcl-2 or Bax. CONCLUSION: Homocysteine-thiolactone enhances apoptosis in cultured human trophoblast, and the effect can be limited by antioxidants.

Apoptosis↗

PubMed2004

Insulin enhances the transcription of luteinizing hormone-beta gene.

OBJECTIVE: Hyperinsulinemia and insulin resistance are implicated in the pathophysiology of polycystic ovary syndrome, a condition associated with elevated levels of LH. We tested the hypothesis that insulin enhances the transcriptional activity of LHbeta promoter. STUDY DESIGN: We transfected the gonadotrope cell line LbetaT2 with a plasmid that expresses the proximal promoter of LHbeta gene upstream of luciferase, and determined insulin effect on endogenous LHbeta mRNA. RESULTS: We found that insulin stimulated (2-4 fold) the activity of this promoter in a time- and concentration-dependent manner. Accordingly, insulin up-regulated the level of LHbeta mRNA. In contrast, insulin had no significant effect on GnRH-dependent LHbeta expression. The expression of Egr-1 and SF-1, which are essential for transcription of LHbeta gene, was unchanged by insulin. CONCLUSION: Insulin enhances the transcription of LHbeta gene. This modulation may contribute to the pathophysiology of polycystic ovary syndrome.

Animals↗

PubMed2004

Is hypotonic dysfunctional labor associated with hypophosphatemia?

OBJECTIVE: The purpose of this study was to determine whether hypotonic dysfunctional labor is associated with hypophosphatemia. STUDY DESIGN: We conducted a prospective nested case control study of term women who were in active labor. Serum phosphate samples were drawn at admission with active labor and before delivery. Phosphate concentration was compared between control subjects and women with hypotonic, dysfunctional uterine contractions. RESULTS: Both serum samples were available for 90 women. Hypophosphatemia was documented in 14% of the participants. There was no significant difference in phosphate concentration between the 2 groups either at admission or before delivery. The mean decrease in phosphate concentration between admission and delivery was similar between the 2 cohorts. CONCLUSION: Although transient hypophosphatemia is found commonly in laboring women, hypotonic dysfunctional contractions are not associated with mild hypophosphatemia.

Adult↗

PubMed2004

Troglitazone attenuates hypoxia-induced injury in cultured term human trophoblasts.

OBJECTIVE: The purpose of this study was to test the hypothesis that the thiazolidinedione troglitazone, a peroxisome proliferator activated receptor-gamma ligand, attenuates hypoxia-induced trophoblast injury. STUDY DESIGN: Cytotrophoblasts from 4 term human placentas were cultured in the presence or absence of 10 mumol/L troglitazone in either 20% oxygen (standard conditions) or 1% oxygen (hypoxic conditions) for variable periods before cell harvest. Medium beta-human chorionic gonadotropin and human placental lactogen were analyzed by enzyme-linked immunosorbent assay. Apoptosis was quantified by cytokeratin-18 cleavage products staining; p53 expression was examined by Western blot analysis. RESULTS: beta-human chorionic gonadotropin and human placental lactogen levels were >/=2-fold higher in troglitazone-exposed cells at 16 hours of hypoxia, compared with vehicle control cells ( P <.05). The apoptotic index was reduced by >/=30% ( P <.001), and the expression of p53 was 2-fold lower ( P <.02) in troglitazone-exposed cells under hypoxia for </=16 hours but not different after >24 hours of low oxygen. CONCLUSION: Troglitazone attenuates the influence of acute hypoxia on cultured term human trophoblasts.

Apoptosis↗

PubMed2004

National Institute on Drug Abuse Conference report on placental proteins, drug transport, and fetal development.

The use of illicit and licit drugs during pregnancy is a major public health concern because of potential adverse effects on the fetus and the risk to maternal health. Because the placenta is the primary link between the mother and the conceptus and is essential for the growth and survival of the fetus, abnormalities in placental formation and function resulting from drug use could have a major influence on pregnancy outcome. At present, little information is available on the impact of abused drugs on placental biology alone or in combination with other "host" factors (eg, stress, infections). This prompted the National Institute on Drug Abuse (NIDA) to convene a meeting of experts in placental biology to review cutting-edge research with the mission to translate existing information to new clinical and research initiatives in the drug abuse field. This report summarizes the presentations and research recommendations resulting from the workshop discussions.

Female↗

PubMed2004

Myocytes of chorionic vessels from placentas with meconium-associated vascular necrosis exhibit apoptotic markers.

Meconium-associated vascular necrosis (MAVN) is a histological abnormality of human placental chorionic vessels that is associated with poor neonatal outcome. We tested the hypothesis that MAVN shows apoptosis in the walls of chorionic vessels. Archival placental specimens with MAVN (n = 5) were compared with specimens from uncomplicated pregnancies at term (n = 5) and from placentas with intense chorionic vasculitis associated with acute chorioamnionitis with (n = 5) or without (n = 5) a clinical history of meconium in the amniotic fluid. Sections from all placentas were processed by the TUNEL method, and 2 observers who were blinded to specimen diagnosis quantified the immunofluorescent TUNEL staining in both the amnion-facing and villous-facing walls of the larger chorionic vessels in each specimen. Compared with the other 3 groups, only the amnion-facing wall of chorionic vessels in MAVN showed a significantly greater number of apoptotic cells. This was verified by morphological criteria and caspase 3 staining. There were limited or no detectable TUNEL-stained cells in either the villous-facing walls of vessels in the MAVN specimens or in any of the vessels of the placentas from uncomplicated pregnancies. There was a negligible level of apoptosis in chorionic vessels of placentas with intense chorionic vasculitis, with or without meconium, despite the inflammatory response or presence of meconium. We conclude that apoptosis contributes to the pathophysiology of MAVN.

Apoptosis↗

PubMed2003

p300 regulates the synergy of steroidogenic factor-1 and early growth response-1 in activating luteinizing hormone-beta subunit gene.

Tight regulation of luteinizing hormone-beta subunit (LHbeta) expression is critical for differentiation and maturation of mammalian sexual organs and reproductive function. Two transcription factors, steroidogenic factor-1 (SF-1) and early growth response-1 (Egr-1), play a central role in activating LHbeta promoter, and the synergy between these two factors is essential in mediating gonadotropin-releasing hormone stimulation of LHbeta promoter. Here we demonstrate that the transcriptional co-activator p300 regulates this synergy. Overexpression of p300 results in strong stimulation of LHbeta promoter but only in the presence of both SF-1 and Egr-1, and not in the presence of other Egr proteins. Mutation of the binding sites for either SF-1 or Egr-1 completely abolishes the synergy between these two factors, as well as the influence of p300. Importantly, LHbeta promoter is precipitated using p300 antibodies in a chromatin immunoprecipitation assay with LbetaT2 gonadotropes, and this effect is enhanced by gonadotropin-releasing hormone. The influence of p300 on LHbeta promoter is potentiated by steroid receptor co-activator, as well as by E1A proteins, and attenuated by Smad nuclear interacting protein 1. Taken together, these results suggest that p300 is recruited to LHbeta promoter where it coordinates the functional synergy between SF-1 and Egr-1.

Adenovirus E1A Proteins↗

PubMed2003

Incorporation of gene-specific variability improves expression analysis using high-density DNA microarrays.

BACKGROUND: The assessment of data reproducibility is essential for application of microarray technology to exploration of biological pathways and disease states. Technical variability in data analysis largely depends on signal intensity. Within that context, the reproducibility of individual probe sets has not been hitherto addressed. RESULTS: We used an extraordinarily large replicate data set derived from human placental trophoblast to analyze probe-specific contribution to variability of gene expression. We found that signal variability, in addition to being signal-intensity dependant, is probe set-specific. Importantly, we developed a novel method to quantify the contribution of this probe set-specific variability. Furthermore, we devised a formula that incorporates a priori-computed, replicate-based information on probe set- and intensity-specific variability in determination of expression changes even without technical replicates. CONCLUSION: The strategy of incorporating probe set-specific variability is superior to analysis based on arbitrary fold-change thresholds. We recommend its incorporation to any computation of gene expression changes using high-density DNA microarrays. A Java application implementing our T-score is available at http://www.sadovsky.wustl.edu/tscore.html.

Algorithms↗

PubMed2003

Microarray analysis of uterine gene expression in mouse and human pregnancy.

Improved care of infants born prematurely has increased their survival. However, the incidence of preterm labor has not changed. To understand the processes involved in preterm labor, we used oligonucleotide microarrays to study gene expression in murine and human uterus during pregnancy. The induction of enzymes for prostaglandin synthesis was used as a marker for important changes during pregnancy because prostaglandins strongly contribute to both human and murine labor. We identified 504 genes that changed at least 2-fold between d 13.5 and 19.0 in the gravid mouse uterus. In the pregnant human myometrium, we found 478 genes that changed at least 2-fold in either term or preterm labor compared with preterm nonlabor specimens and 77 genes that significantly varied in both preterm and term labor. Patterns of gene regulation within functional groups comparing human preterm and term labor were similar, although the magnitude of change often varied. Surprisingly, few genes that changed significantly throughout pregnancy were the same in the mouse and human. These data suggest that functional progesterone withdrawal in human myometrium may not be the primary mechanism for labor induction, may implicate similar mechanisms for idiopathic preterm and term labor in humans, and may identify novel targets for further study.

Animals↗

PubMed2003

Human placenta metabolizes fatty acids: implications for fetal fatty acid oxidation disorders and maternal liver diseases.

The role of fat metabolism during human pregnancy and in placental growth and function is poorly understood. Mitochondrial fatty acid oxidation disorders in an affected fetus are associated with maternal diseases of pregnancy, including preeclampsia, acute fatty liver of pregnancy, and the hemolysis, elevated liver enzymes, and low platelets syndrome called HELLP. We have investigated the developmental expression and activity of six fatty acid beta-oxidation enzymes at various gestational-age human placentas. Placental specimens exhibited abundant expression of all six enzymes, as assessed by immunohistochemical and immunoblot analyses, with greater staining in syncytiotrophoblasts compared with other placental cell types. beta-Oxidation enzyme activities in placental tissues were higher early in gestation and lower near term. Trophoblast cells in culture oxidized tritium-labeled palmitate and myristate in substantial amounts, indicating that the human placenta utilizes fatty acids as a significant metabolic fuel. Thus human placenta derives energy from fatty acid oxidation, providing a potential explanation for the association of fetal fatty acid oxidation disorders with maternal liver diseases in pregnancy.

Adult↗

PubMed2003

A novel domain within the DEAD-box protein DP103 is essential for transcriptional repression and helicase activity.

Members of the DEAD-box family of helicases, distinguished by a core characteristic sequence of Asp-Glu-Ala-Asp, are expressed in a wide range of prokaryotes and eukaryotes and exhibit diverse cellular functions, including DNA transcription, recombination and repair, RNA processing, translation, and posttranslational regulation. Although ubiquitous, the function of most DEAD-box proteins is unknown. We and others have recently cloned DP103, which harbors conserved DEAD-box, helicase, and ATPase domains in its N terminus. DP103 (also termed Gemin3 and DDX20) interacts with SF-1, SMN, EBNA2, and EBNA3C in mammalian cells. Here we demonstrate that a discrete domain within the nonconserved C-terminal region of DP103 directly interacts with SF-1. This domain exhibits an autonomous repression function and is necessary and sufficient for repressing the transcriptional activity of SF-1. Furthermore, intact DP103 exhibits helicase activity. Importantly, the C-terminal domain is obligatory but not sufficient for this unwinding activity of DP103. Together, our results support a novel paradigm for transcriptional repression and demonstrate the bifunctional role of the C-terminal domain of DP103.

Animals↗

PubMed2003

The lipid droplet-associated protein adipophilin is expressed in human trophoblasts and is regulated by peroxisomal proliferator-activated receptor-gamma/retinoid X receptor.

Uptake and transplacental transfer of fatty acids is essential for fetal development. Human adipophilin and its murine ortholog adipocyte differentiation-related protein are lipid droplet-associated proteins that are implicated in cellular fatty acid uptake in adipocytes. The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) stimulates lipid uptake by adipocytes and enhances differentiation of placental trophoblasts. We therefore hypothesized that adipophilin is expressed in human trophoblasts and that its expression is regulated by PPARgamma. We initially determined that adipophilin is expressed in human villous trophoblasts and that adipophilin expression is enhanced during differentiation of cultured primary term human trophoblasts. We also found that exposure of cultured human trophoblasts to the PPARgamma ligand troglitazone resulted in a concentration-dependent increase in adipophilin expression. We observed a similar increase with LG268, a ligand for retinoid X receptor (RXR), the heterodimeric partner of PPARgamma. Lastly, we demonstrated that ligand-activated PPARgamma and RXR stimulated the transcriptional activity of adipophilin promoter in CV-1 cells and in the placental JEG3 cell line. We conclude that the expression of adipophilin is enhanced during trophoblast differentiation and is up-regulated by ligand-activated PPARgamma/RXR. Enhanced adipophilin expression may contribute to fatty acid uptake by the placenta.

Anticholesteremic Agents↗

PubMed2002

A variable fold change threshold determines significance for expression microarrays.

The use of expression microarrays to determine bona fide changes in gene expression between experimental paradigms is confounded by noise due to variability in measurement. To assess the variability associated with transcript hybridization to commercial oligonucleotide-based microarrays, we generated a data set consisting of five replicate hybridizations of a single labeled cRNA target from three distinct experimental paradigms, using the Affymetrix human U95 GeneChip set. We found that the variability of expression level in our data set is intensity-specific. We quantified the observed variability in our data set in order to determine significant changes in gene expression. LOESS fitting to a plot of the standard deviation of replicates assigned a variability associated with a specific intensity. This allowed for the calculation of a "variable fold-change" threshold for any absolute intensity at any level of statistical confidence. Testing of this method indicates that it removes intensity-specific bias and results in a 5- to 10-fold reduction in the number of false-positive changes. We suggest that this approach can be widely used to improve prediction of significant changes in gene expression for oligonucleotide-based microarray experiments and reduce false leads, even in the absence of replicates.

Gene Expression Profiling↗

PubMed2002

Trophoblast apoptosis from pregnancies complicated by fetal growth restriction is associated with enhanced p53 expression.

OBJECTIVE: We tested the hypothesis that apoptotic trophoblasts from pregnancies associated with fetal growth restriction caused by preeclampsia or cigarette use exhibit enhanced expression of the proapoptotic proteins p53 and Bax and diminished expression of the antiapoptotic protein Bcl-2. STUDY DESIGN: Placentas were obtained from women with uncomplicated pregnancies (n = 4) or from women with pregnancies complicated by fetal growth restriction associated with preeclampsia, cigarette use, or both (n = 7). Placental sections were examined by means of hematoxylin and eosin and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining, as well as by detection of cytokeratin 18 cleavage products indicative of apoptosis. The expression of p53 was examined by means of Western immunoblotting and immunohistochemistry. The expression of Bax, Bcl-2, Bak, and Bcl-X(L) was analyzed by immunoblotting. RESULTS: More apoptosis was found in the trophoblast layer of villi from pregnancies complicated by fetal growth restriction than in the trophoblast layer of villi from control pregnancies. The enhanced apoptosis correlated with up-regulation of p53, primarily in cytotrophoblast nuclei. There was no difference between the two groups in expression of the proteins from the Bcl-2 family. CONCLUSIONS: The expression of p53, but not members of the Bcl-2 family of proteins is up-regulated in human placental villi from pregnancies complicated by fetal growth restriction. We speculate that conditions predisposing to placental hypoxia lead to p53-mediated apoptosis in trophoblasts and thereby contribute to placental dysfunction.

Adult↗

PubMed2002

A prospective randomized safety trial of celecoxib for treatment of preterm labor.

OBJECTIVE: We compared the safety of celecoxib, a selective cyclo-oxygenase-2 inhibitor, with the safety of the nonselective cyclo-oxygenase inhibitor indomethacin, when it was administered for treatment of preterm labor. STUDY DESIGN: In a randomized, double-blind, placebo-controlled trial, 24 pregnant women in preterm labor at 24 to 34 weeks of gestation received either indomethacin or celecoxib for 48 hours. Clinical assessment, fetal sonography, and Doppler blood flow studies of the fetal ductus arteriosus were performed daily. RESULTS: Mean maximum ductal flow velocity was significantly elevated over baseline (82.9 +/- 4.6 cm/s vs 111.14 +/- 14.3 cm/s; P =.02) after 24 hours of indomethacin, but not celecoxib. Both medications were associated with a transient decrease in amniotic fluid volume, with a greater effect by indomethacin. The medications were equally effective in the maintenance of tocolysis. There were no significant maternal or neonatal adverse events. CONCLUSION: In this initial evaluation, the safety of short-term celecoxib in women with preterm labor was superior to that of indomethacin.

Adolescent↗

PubMed2002

Trophoblast differentiation modulates the activity of caspases in primary cultures of term human trophoblasts.

Cultured human cytotrophoblasts are more susceptible than syncytiotrophoblasts to hypoxia-induced apoptosis. Caspases are cysteine proteases that cleave cellular components to effect the apoptotic cascade. We hypothesized that cultured cytotrophoblasts exhibit a higher activity of caspases when compared with syncytiotrophoblasts. Using western analysis, we demonstrated that the pro-caspases 3, 6, 8, and 9 are expressed in cytotrophoblasts cultured for 24 h, and also, in trophoblasts cultured 72 h when syncytiotrophoblasts have formed. Importantly, we found significantly higher activity of all four caspases in trophoblasts cultured 24 h compared with cells cultured 72 h. Colchicine and DMSO, which hinder trophoblast differentiation, enhanced the activity of all four caspases in cells cultured 72 h. Conversely, caspase activity was reduced in trophoblasts cultured for 24 h in the presence of epidermal growth factor, which enhances differentiation. This effect was most pronounced on caspase 3 and was attenuated by addition of the tyrosine kinase inhibitor AG1478. We conclude that cytotrophoblasts exhibit a higher activity of caspases 3, 6, 8, and 9 when compared with the more differentiated syncytium. This may account for the higher susceptibility of cytotrophoblasts to hypoxia-induced apoptosis.

Amino Acid Chloromethyl Ketones↗