High-fat diet in pregnant rats and adverse fetal outcome.

Background: Although pregestational obesity has been associated with increased risk of adverse fetal outcome, the mechanisms behind are not known. We aimed to investigate the influence of the maternal metabolic state on fetal outcome in rats exposed to either a high-fat diet (HFD) or a control diet (CD). We also investigated the impact of serum collected from HFD/CD pregnant rats on CD embryonic development in whole-embryo cultures. Material and methods: On gestational day 0, 9, 10, or 20 maternal plasma/serum samples were collected as pregnancies were terminated for the estimations of maternal metabolic state and embryo-fetal development. We measured embryonic gene expression of ROS scavenger enzymes as well as genes involved in inflammation in maternal adipose tissue. Results: In HFD maternal plasma/serum, concentrations of glucose, ß-hydroxybutyrate, branched-chain amino acids, and leptin were increased, whereas those of triacylglycerol, cholesterol, and palmitic, oleic, linoleic, and α-linolenic acids were decreased. Gene expression of CuZnSOD, IL-6, IL-10, and resistin was increased in HFD maternal adipose tissue, whereas that of CuZnSOD and MnSOD was decreased in HFD-exposed embryos. HFD caused retention of most fatty acids in the maternal liver as well. Conclusion: HFD alters the maternal metabolic state, increases fetal resorptions in vivo, and increases the rate of fetal/embryonic malformations both in vivo and in vitro. These findings suggest that metabolic disturbances in HFD pregnant rats have profound adverse developmental effects in the offspring.

The status of diabetic embryopathy.

Diabetic embryopathy is a theoretical enigma and a clinical challenge. Both type 1 and type 2 diabetic pregnancy carry a significant risk for fetal maldevelopment, and the precise reasons for the diabetes-induced teratogenicity are not clearly identified. The experimental work in this field has revealed a partial, however complex, answer to the teratological question, and we will review some of the latest suggestions.

Diabetic embryopathy.

Diabetic embryopathy reflects a scientific enigma--how does a seemingly rich intrauterine environment manage to disturb the development of the embryo? Which compounds in that environment may be teratogenic--and how shall we find them? How can we investigate a putative dose-response nature of the teratogen, i.e., how can we monitor the effects of varied severity of the diabetic state (which can be varied in a number of metabolic ways) on the embryonic development? Here, the whole embryo culture (WEC) technique provides an excellent tool for such studies. WEC is thus currently used to investigate the effect of graded levels of diabetes (e.g., hyperglycemia, hyperketonemia, increased branched chain amino acid (BCAA) levels), and putative antiteratogenic agents (antioxidants, folic acid, arachidonic acid, inositol), as well as the effect of different embryonic genotypes on diabetes-induced (mal)development. WEC is the only method, which is able to couple specific embryonic maldevelopment to precise changes in substrate levels or the (epi)genotype of the embryo. Using this method, we have been able to demonstrate that a diabetic environment--culture of embryos in serum from diabetic animals or in serum with increased levels of glucose, ß-hydroxybutyrate or α-ketoisocaproic acid (KIC)--causes increased embryonic maldevelopment, and that this dysmorphogenesis is blocked by the addition of ROS scavenging agents to the culture medium. Genetically, others and we have demonstrated that Pax-3 downregulation predisposes for diabetes-induced dysmorphogenesis.

Altered gene expression in rat cranial neural crest cells exposed to a teratogenic glucose concentration in vitro: paradoxical downregulation of antioxidative defense genes.

BACKGROUND: Diabetic pregnancy is associated with increased risk of malformation in the infant. Diabetes-induced anomalies of the face and heart are strongly correlated with neural crest cell (NCC) maldevelopment. We aimed to study glucose-induced alterations of mRNA levels in cranial and trunk NCCs isolated from rat embryos with increased risk of developing mandibular and cardiac malformations in diabetic pregnancy. METHODS: Inbred Sprague-Dawley rat embryos were used for NCC isolation from neural tube explants. The migrating cells were exposed to 5.5 or 30 mmol/l glucose concentration for 48 hr, harvested, and prepared for gene expression measurement by RT-PCR or immunostaining with either distal-less (Dlx) or AP-2-α antibodies. RESULTS: Evaluation of the immunostained slides showed that approximately 75% of the cells were of NCC origin. Exposure to 30 mM glucose decreased mRNA levels of Copper-Zinc superoxide dismutase, manganese superoxide dismutase, extracellular superoxide dismutase, Catalase, Gpx-1, Nrf2, poly-ADP ribose polymerase, B-cell leukemia/lymphoma protein 2, and ß-Catenin genes in cranial neural crest explant cultures. In addition, Pax-3, Pax-6, Wnt3a, and Apc mRNA levels were decreased by high glucose exposure in both cranial and trunk neural crest explant cultures. CONCLUSION: Cranial NCCs diminish their mRNA levels of antioxidative enzymes and the Nrf2 response factor, as well as the antiapoptotic B-cell leukemia/lymphoma protein 2 gene, in response to increased ambient glucose concentration. Furthermore, both cranial and trunk NCC decrease the mRNA levels of the transcription factors Pax-3 and Pax-6, as well as key components of the Wnt pathway. These patterns of glucose-altered gene expression in a developmentally important cell population may be of etiological importance for NCC-associated malformations in diabetic pregnancy.

Prodynorphin CpG-SNPs associated with alcohol dependence: elevated methylation in the brain of human alcoholics.

The genetic, epigenetic and environmental factors may influence the risk for neuropsychiatric disease through their effects on gene transcription. Mechanistically, these effects may be integrated through regulation of methylation of CpG dinucleotides overlapping with single-nucleotide polymorphisms (SNPs) associated with a disorder. We addressed this hypothesis by analyzing methylation of prodynorphin (PDYN) CpG-SNPs associated with alcohol dependence, in human alcoholics. Postmortem specimens of the dorsolateral prefrontal cortex (dl-PFC) involved in cognitive control of addictive behavior were obtained from 14 alcohol-dependent and 14 control subjects. Methylation was measured by pyrosequencing after bisulfite treatment of DNA. DNA binding proteins were analyzed by electromobility shift assay. Three PDYN CpG-SNPs associated with alcoholism were found to be differently methylated in the human brain. In the dl-PFC of alcoholics, methylation levels of the C, non-risk variant of 3'-untranslated region (3'-UTR) SNP (rs2235749; C > T) were increased, and positively correlated with dynorphins. A DNA-binding factor that differentially targeted the T, risk allele and methylated and unmethylated C allele of this SNP was identified in the brain. The findings suggest a causal link between alcoholism-associated PDYN 3'-UTR CpG-SNP methylation, activation of PDYN transcription and vulnerability of individuals with the C, non-risk allele(s) to develop alcohol dependence.

Altered gene expression in neural crest cells exposed to ethanol in vitro.

AIM: to characterize and compare ethanol-induced changes of gene expression in cells from the cranial (cNCC) and trunk (tNCC) portion of the neural crest cell (NCC) population of day-10 rat embryos. BACKGROUND: previous work has suggested that ethanol-induced embryonic maldevelopment is associated with oxidative stress, and, in particular, that ethanol-induced anomalies of the facial skeleton and heart are associated with disturbed development of the cNCC. We studied alterations of mRNA levels of genes involved in apoptosis, oxidative defense, cellular metabolism, NCC development or inflammation in cNCC and tNCC from rat embryos exposed to ethanol in vitro. We specifically evaluated expression differences between cNCC and tNCC genes, possibly reflecting the different teratological susceptibilities of the two cell populations. METHODS: neural tube explants from rat embryos were divided in cranial and trunk portions and used for NCC isolation in vitro on gestational day 10. The migrating cells from the cranial or trunk explants of the neural tube were subsequently exposed to 0 or 88 mmol/l ethanol concentration with or without addition of 0.5 mM N-acetylcysteine (NAC) for 48 h, harvested, and prepared for gene expression measurement by RT-PCR or immunostaining with either distal-less (DLX) or AP 2-alpha antibodies. RESULTS: evaluation of the immunostained slides showed that approximately 75% of the cNCC and tNCC preparations were of neural crest origin. Exposure to 88 mM ethanol increased the Bax/Bcl-2 ratio in the NCC, and NAC addition diminished this increase. Both cNCC and tNCC upregulated MnSOD and Gpx-1 in response to ethanol, whereas tNCC increased CuZnSOD and EC-SOD after ethanol exposure (cNCC unchanged). Expression of glyceraldehyde-3-phosphate dehydrogenase was downregulated by ethanol in cNCC only. In addition, ethanol exposure caused increased mRNA levels of Pax-3, p53, Vegf-A and decreased expression of Pax-6, Nfe2 in both cNCC and tNCC. Ethanol increased Shh and Bmp-4 and decreased Parp only in cNCC (tNCC unchanged), whereas ethanol exposure increased T box-2 and decreased Gdnf and Ret only in tNCC (cNCC unchanged). In addition, ethanol exposure almost abolished expression of Hox a(1), a(4) and a(5), and left Hox a(2) unchanged in cNCC, whereas all four of these Hox genes were upregulated in tNCC. CONCLUSIONS: ethanol causes a shift towards apoptosis in both cNCC and tNCC, a shift, which is diminished by NAC treatment. Oxidative defense genes, and genes involved in neural crest cell development are affected differently in cNCC compared to tNCC upon ethanol exposure. Moreover, ethanol downregulates cNCC Hox genes, whereas tNCC Hox genes are upregulated. These patterns of ethanol-altered gene expression may be of etiological importance for NCC-associated maldevelopment in ethanol-exposed pregnancy.

Angiogenesis inhibition causes hypertension and placental dysfunction in a rat model of preeclampsia.

BACKGROUND: Preeclampsia is a serious pregnancy complication, accompanied by increased maternal and fetal morbidity. Different models have been used to study preeclampsia, but none of these display all the key features of the disease. METHOD: We investigated the effects on maternal blood pressure and fetal outcome exerted by the angiogenesis inhibitor Suramin (100 mg/kg i.p.) during early placentation. Blood pressure and heart rate were measured continuously with telemetry in Sprague-Dawley rats of four experimental groups: nonpregnant controls, Suramin-treated nonpregnant rats, pregnant controls and pregnant Suramin-treated rats. Blood samples were collected before pregnancy and at gestational day 20 for analysis of renin and sFlt-1. The fetal and placental morphology were evaluated after caesarian section on gestational day 20. RESULTS: The blood pressure of the pregnant Suramin-treated rats successively increased during pregnancy and differed by 17 mmHg at gestational day 20 compared with the pregnant control rats. In the pregnant Suramin-treated rats group, the renin levels increased (+122%) and the sFlt-1 levels decreased (-58%) during pregnancy. The pregnant Suramin-treated fetuses and placentae were smaller (2.8 g and 0.51 g) than the pregnant controls rats' fetuses and placentae (3.5 g and 0.56 g). Resorptions tended to be higher in the pregnant Suramin-treated rat litters compared with the pregnant control rat litters (P = 0.08). The area of the maternal blood vessels in the mesometrial triangle was smaller in the pregnant Suramin-treated rats group than in the pregnant control rats group. CONCLUSION: The inhibition of uterine angiogenesis increases maternal blood pressure and compromises fetal and placental development. Placental hypoxia and subsequent activation of the renin-angiotensin system may play an important role for the hypertension.

Can we prevent diabetic birth defects with micronutrients?

Congenital malformations are more common in infants of diabetic women than in children of non-diabetic women. The mechanisms behind diabetes-induced congenital anomalies are not known. Disturbed micronutrient metabolism, in concert with oxidative stress, has been suggested as a cause of diabetes-induced malformations by several studies. In experimental work, administration of inositol, arachidonic acid and several antioxidative compounds, as well as folic acid, to the embryo, has proven to attenuate the teratogenic effects of a diabetic environment. Future therapeutic efforts may include supplementation with antioxidants or micronutrients, such as folic acid, to the pregnant diabetic woman, although exact compounds and doses need to be determined.

Specific local cardiovascular changes of Nepsilon-(carboxymethyl)lysine, vascular endothelial growth factor, and Smad2 in the developing embryos coincide with maternal diabetes-induced congenital heart defects.

OBJECTIVE: Embryos exposed to a diabetic environment in utero have an increased risk to develop congenital heart malformations. The mechanism behind the teratogenicity of diabetes still remains enigmatic. Detrimental effects of glycation products in diabetic patients have been well documented. We therefore studied a possible link between glycation products and the development of congenital cardiovascular malformations. Furthermore, we investigated other possible mechanisms involved in this pathogenesis: alterations in the levels of vascular endothelial growth factor (VEGF) or phosphorylated Smad2 (the latter can be induced by both glycation products and VEGF). RESEARCH DESIGN AND METHODS: We examined the temporal spatial patterning of the glycation products Nepsilon(carboxymethyl)lysine (CML) and methylglyoxal (MG) adducts, VEGF expression, and phosphorylated Smad2 during cardiovascular development in embryos from normal and diabetic rats. RESULTS: Maternal diabetes increased the CML accumulation in the areas susceptible to diabetes-induced congenital heart disease, including the outflow tract of the heart and the aortic arch. No MG adducts could be detected, suggesting that CML is more likely to be indicative for increased oxidative stress than for glycation. An increase of CML in the outflow tract of the heart was accompanied by an increase in phosphorylated Smad2, unrelated to VEGF. VEGF showed a time-specific decrease in the outflow tract of embryos from diabetic dams. CONCLUSIONS: From our results, we can conclude that maternal diabetes results in transient and localized alterations in CML, VEGF expression, and Smad2 phosphorylation overlapping with those regions of the developing heart that are most sensitive to diabetes-induced congenital heart disease.

Decreased cardiac glutathione peroxidase levels and enhanced mandibular apoptosis in malformed embryos of diabetic rats.

OBJECTIVE: To characterize normal and malformed embryos within the same litters from control and diabetic rats for expression of genes related to metabolism of reactive oxygen species (ROS) or glucose as well as developmental genes. RESEARCH DESIGN AND METHODS: Embryos from nondiabetic and streptozotocin-induced diabetic rats were collected on gestational day 11 and evaluated for gene expression (PCR) and distribution of activated caspase-3 and glutathione peroxidase (Gpx)-1 by immunohistochemistry. RESULTS: Maternal diabetes (MD group) caused growth retardation and an increased malformation rate in the embryos of MD group rats compared with those of controls (N group). We found decreased gene expression of Gpx-1 and increased expression of vascular endothelial growth factor-A (Vegf-A) in malformed embryos of diabetic rats (MDm group) compared with nonmalformed littermates (MDn group). Alterations of messenger RNA levels of other genes were similar in MDm and MDn embryos. Thus, expression of copper zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), and sonic hedgehog homolog (Shh) were decreased, and bone morphogenetic protein-4 (Bmp-4) was increased, in the MD embryos compared with the N embryos. In MDm embryos, we detected increased activated caspase-3 immunostaining in the first visceral arch and cardiac area and decreased Gpx-1 immunostaining in the cardiac tissue; both findings differed from the caspase/Gpx-1 immunostaining of the MDn and N embryos. CONCLUSIONS: Maternal diabetes causes growth retardation, congenital malformations, and decreased general antioxidative gene expression in the embryo. In particular, enhanced apoptosis of the first visceral arch and heart, together with decreased cardiac Gpx-1 levels, may compromise the mandible and heart and thus cause an increased risk of developing congenital malformation.

Maternal blood glucose levels determine the severity of diabetic embryopathy in mice with different expression of copper-zinc superoxide dismutase (CuZnSOD).

Excess oxygen radical formation is suggested to be involved in the etiology of diabetic embryopathy. We aimed to investigate the effects of altered maternal antioxidative status in conjunction with a varied severity of the maternal diabetic state on embryonic development by using mice with different gene expression of CuZn superoxide dismutase (CuZnSOD). The mice were wild-type (WT), transgenic (TG), or knockout (KO) with regard to CuZnSOD. Alloxan was used to induce diabetes (DWT, DTG, DKO) in female mice before pregnancy and, noninjected mice served as controls (NWT, NTG, NKO). The minimum alloxan dose required to induce diabetes was 80 mg/kg for WT, 100 mg/kg for TG, and 65 mg/kg for KO mice. When KO mice were made diabetic with 80 mg/kg alloxan, they produced no living offspring. The pregnancies were interrupted on gestational day 18, when maternal diabetic state, that is, blood glucose concentration, as well as fetal outcome, genotype and hepatic isoprostane levels were assessed. The mean maternal blood glucose levels were positively associated with the alloxan dose, that is, the DWT and DTG groups had higher blood glucose concentration than the DKO group, and the DWT and DTG fetuses increased their hepatic isoprostane levels, whereas the DKO fetuses did not. However, in all diabetic groups, increased maternal blood glucose concentration was associated with higher resorption and malformation rates as well as lowered fetal and placental weight. Furthermore, diabetes increased the fraction of WT offspring in the TG and KO groups. We conclude that both fetal antioxidative capacity and maternal diabetic state affect the development of the offspring. However, the maternal diabetic state is the major teratogenic factor and overrides the influence of fetal antioxidative capacity.

Genetic influence on dysmorphogenesis in embryos from different rat strains exposed to ethanol in vivo and in vitro.

BACKGROUND: The aim was to investigate the susceptibility of embryos from 2 rat strains (U and H) to a 48 hours ethanol exposure in early pregnancy, both in vivo and in vitro. METHODS: The embryos were studied on gestational days 9 to 11. We used 1 ethanol dose in vivo (6 g/kg x 2), 3 different ethanol concentrations in vitro (88 mM, 132 mM, 176 mM) and also attempted to diminish the teratogenic effect in vitro by supplying the antioxidant N-acetylcysteine (NAC, 0.5 mM) to the culture medium. RESULTS: The U embryos were more damaged by ethanol than the H embryos, both in vivo and in vitro. NAC addition diminished, but failed to completely normalize, the embryonic maldevelopment. Ethanol increased the Bax/Bcl-2 ratio in the U embryos both in vivo and in vitro, but not in the H embryos. Furthermore, ethanol caused increased Caspase-3 immunostaining in U embryos, but not in H embryos. Ethanol exposure in vivo did not alter CuZnSOD and MnSOD mRNA levels in U and H embryos. In vitro, however, the ethanol-exposed U embryos increased their CuZnSOD and MnSOD mRNA levels, whereas the CuZnSOD mRNA was unchanged and MnSOD mRNA decreased in the H embryos, in neither strain did NAC exert any effect. The U embryos increased catalase gene expression in response to ethanol in vivo, but decreased catalase mRNA levels in vitro, changes normalized by NAC. The H embryos did not alter catalase mRNA levels in vivo, but increased gene expression in vitro, with no NAC effect. Ethanol affected the gene expression of the other ROS scavenging enzymes and the developmental genes studied - Bmp-4, Ret, Shh, Pax-6 - similarly in the 2 strains. CONCLUSIONS: The findings support a role for genetic predisposition, oxidative stress, and apoptosis in ethanol teratogenicity, and suggest that the teratogenic predisposition of the more susceptible U rats may reside, at least in part, in the regulation of the ROS scavenging enzymes in the U embryos.

Shb null allele is inherited with a transmission ratio distortion and causes reduced viability in utero.

SHB is an Src homology 2 domain-containing adapter protein that has been found to be involved in numerous cellular responses. We have generated an Shb knockout mouse. No Shb-/- pups or embryos were obtained on the C57Bl6 background, indicating an early defect as a consequence of Shb- gene inactivation on this genetic background. Breeding heterozygotes for Shb gene inactivation (Shb+/-) on a mixed genetic background (FVB/C57Bl6/129Sv) reveals a distorted transmission ratio of the null allele with reduced numbers of Shb+/+ and Shb-/- animals, but increased number of Shb+/- animals. The Shb- allele is associated with various forms of malformations, explaining the relative reduction in the number of Shb-/- offspring. Shb-/- animals that were born were viable, fertile, and showed no obvious defects. However, Shb+/- female mice ovulated preferentially Shb- oocytes explaining the reduced frequency of Shb+/+ mice. Our study suggests a role of SHB during reproduction and development.

Folic acid supplementation affects ROS scavenging enzymes, enhances Vegf-A, and diminishes apoptotic state in yolk sacs of embryos of diabetic rats.

We aimed to investigate the extent to which maternal diabetes with or without folic acid (FA) supplementation affects mRNA levels and protein distribution of ROS scavenging enzymes, vascular endothelial growth factor-A (Vegf-A), folate binding protein-1 (Folbp-1), and apoptosis-associated proteins in the yolk sacs of rat embryos on gestational days 10 and 11. Commencing at conception and throughout pregnancy, half of the streptozotocin-diabetic and half of the control rats received daily FA injections. Maternal diabetes impaired vascular morphology and decreased CuZnSOD and GPX-1 gene expression in yolk sacs. Maternal diabetes also increased the levels of CuZnSOD protein, increased the Bax/Bcl-2 protein ratio and decreased Vegf-A protein distribution. FA treatment normalized vascular morphology, decreased mRNA levels of all three SOD isoforms and increased Vegf-A mRNA levels, rectified CuZnSOD protein distribution and Bax/Bcl-2 ratio. A teratogenic diabetic environment produces a state of vasculopathy, oxidative stress, and mild apoptosis in the yolk sac. FA administration normalizes vascular morphology, diminishes apoptotic rate, and increases Vegf-A gene expression and protein distribution in the yolk sac of diabetic rats.

N-Acetylcysteine and alpha-cyano-4-hydroxycinnamic acid alter protein kinase C (PKC)-delta and PKC-zeta and diminish dysmorphogenesis in rat embryos cultured with high glucose in vitro.

Malformations and growth disturbances are two- to threefold more common in infants of diabetic mothers than in offspring of non-diabetic pregnancy. Several suggestions have emerged to explain the reasons for diabetic embryopathy, including enhanced mitochondrial production of reactive oxygen species leading to altered activation of protein kinase C. This study aimed to evaluate the effect of alpha-cyano-4-hydroxycinnamic acid (CHC) and N-acetylcysteine (NAC) addition on morphology and activity of protein kinase C-delta and protein kinase C-zeta in rat embryos exposed to a high glucose concentration in vitro. Day 9 embryos from normal rats were cultured in 10 or 30 mM glucose concentrations with or without supplementation of CHC, NAC, or protein kinase C inhibitors specific for protein kinase C-delta and protein kinase C-zeta. Embryos were evaluated for malformations, crown rump length, and somite number. Protein kinase C-delta and protein kinase C-zeta activities were estimated by western blot by separating membranous and cytosolic fractions of the embryo. We found increased malformations and growth retardation in embryos cultured in high versus low glucose concentrations. These abnormalities were diminished when CHC and NAC or specific protein kinase C-inhibitors were added to the culture medium. The activities of embryonic protein kinase C-delta and protein kinase C-zeta were increased in the high glucose environment after 24-h culture, but were normalized by the addition of CHC and NAC as well as respective inhibitor to the culture medium. These findings suggest that mitochondrial overproduction of reactive oxygen species is involved in diabetic embryopathy. Furthermore, such overproduction may affect embryonic development, at least partly, by enhancing the activities of protein kinase C-delta and protein kinase C-zeta.

Maternal diabetes in vivo and high glucose concentration in vitro increases apoptosis in rat embryos.

Apoptosis may be involved in diabetes-induced embryonic dysmorphogenesis. We estimated the occurrence of apoptosis in embryos of a rat model for diabetic pregnancy. We found decreased Bcl-2, increased Bax and cleaved Caspase 3 proteins in embryos from diabetic rats. Moreover, we found increased activation of Caspase 3 in cells from embryos previously exposed to a diabetes-like environment (in vivo, in vitro) compared to cells from control embryos, which was normalized by supplementation of N-acetylcysteine or apoptosis inhibitor. We detected increased propidium iodide uptake in embryonic cells exposed to maternal diabetes, a finding confirmed by vital staining. Additionally, we found increased dysmorphogenesis in embryos exposed to a diabetic environment in vivo and in vitro. Exposure to a diabetic milieu during organogenesis increases apoptosis in embryonic cells and dysmorphogenesis in embryos. Enhanced apoptotic rate may have a role in diabetic embryopathy by inducing disturbed embryonic maturation, increased rates of resorptions and congenital malformations.

Antioxidative treatment diminishes ethanol-induced congenital malformations in the rat.

BACKGROUND: Intrauterine exposure to ethanol causes embryonic and fetal growth retardation and maldevelopment. Oxidative stress in mother and offspring has been suggested to be part of the teratogenic mechanism, and supplementation of antioxidative agents to the pregnant women may therefore be of value in future prophylactic treatment regimen. There is a need for in vivo experimental work in this field, and in the present study, our aim was to investigate whether chronic ethanol consumption induced congenital malformations in rats and, if so, whether dietary supplementation of vitamin E (alpha-tocopherol) diminished such maldevelopment. METHODS: Female Sprague-Dawley rats were given drinking water containing 20% ethanol and half of these received food containing 5% vitamin E. Non-ethanol-exposed female rats, with or without vitamin E treatment, served as controls. The pregnancy was interrupted on gestational day 20 when the offspring was evaluated morphologically and fetal hepatic 8-iso-PGF(2alpha) levels were measured to assess the degree of fetal oxidative stress. RESULTS: Exposure to 20% ethanol increased maternal blood ethanol to 1.5 promille and increased resorption and malformation rates in the offspring. Maternal vitamin E treatment did not affect blood ethanol levels, but normalized fetal development. The fetal hepatic levels of 8-iso-PGF(2alpha) were increased in the ethanol-exposed group and normalized by vitamin E treatment of the mother. CONCLUSIONS: Ethanol exposure disturbs embryogenesis partly by enhanced oxidative stress, and the adverse effects can be ameliorated by antioxidative treatment.

Combined supplementation of folic acid and vitamin E diminishes diabetes-induced embryotoxicity in rats.

BACKGROUND: Oxidative stress and enhanced apoptosis may be involved in the induction of embryonic dysmorphogenesis in diabetic pregnancy. Administration of folic acid or vitamin E diminishes embryonic dysmorphogenesis. We aimed to evaluate the effect of combined treatment with folic acid and vitamin E on the disturbed development in embryos of diabetic rats. METHODS: Pregnant nondiabetic and diabetic rats were treated with daily injections of 15 mg/kg folic acid or with 5% vitamin E in the diet. A third group received combined treatment. Day 10 and day 11 embryos were evaluated for development and apoptotic profile. RESULTS: We found increased malformations, resorptions, and profound growth retardation in embryos of diabetic rats compared to control embryos. Vitamin E or folic acid alone, or the 2 compounds combined, normalized embryonic demise. Maternal diabetes caused decreased nuclear factor-kappaB (NF-kappaB) activity and B-cell lymphoma 2 (Bcl-2) protein level, and increased Bcl-2-associated x proteins (Bax) in embryos. Supplementation of vitamin E alone normalized the Bax protein level in a diabetic environment. Administration of folic acid to diabetic rats increased NF-kappaB activity and Bcl-2 protein level. Combined treatment normalized Bcl-2 and Bax protein level in a diabetic environment. CONCLUSIONS: Combined supplementation of folic acid and vitamin E to pregnant diabetic rats diminished diabetes-induced malformations and resorptions, concomitant with normalization of apoptotic protein levels. No treatment completely abolished the embryonic demise; therefore, other mechanisms than oxidative stress and apoptosis are likely to be involved in diabetic embryopathy.

Ethanol-induced fetal dysmorphogenesis in the mouse is diminished by high antioxidative capacity of the mother.

Intrauterine exposure to ethanol causes embryonic and fetal maldevelopment. Oxidative stress in mother and offspring has been suggested to be part of the teratogenic mechanism of ethanol. Here we aimed to assess the importance of maternal and fetal antioxidative capability for the risk of dysmorphogenesis in the offspring. We used male and female mice with different levels of superoxide dismutase (SOD) activity-wild-type (WT) mice, mice with a targeted SOD mutation (KO, decreased CuZnSOD mRNA), and mice transgenic for SOD (TG, increased CuZnSOD mRNA). Female WT, KO (heterozygous), and TG (heterozygous) mice were given drinking water containing 20% ethanol before and throughout gestation. Non-ethanol-exposed WT, KO, and TG mice served as controls. The female mice were mated with males with identical genotype, and the pregnancy was interrupted on gestational day 18 when the offspring was evaluated and genotyped. Fetal hepatic isoprostane (8-epi-PGF(2alpha)) levels were measured to assess the degree of fetal oxidative stress. Exposure to 20% ethanol decreased fetal weight by 9-13% in the three groups. Ethanol exposure roughly doubled the rates of maldeveloped WT and KO offspring but did not affect TG offspring. The fetal hepatic levels of 8-epi-PGF(2alpha) were increased in the ethanol-exposed WT and KO mice but not in ethanol-exposed TG mice. Ethanol exposure preferentially damaged WT fetuses in pregnant KO mice, whereas no such effect was found in the litters of ethanol-consuming TG mice. Administration of ethanol to pregnant mice disturbs embryogenesis by oxidative stress, and the adverse effects are more pronounced in offspring of mice with low antioxidative capacity.

Platelet-derived growth factor receptor-beta promotes early endothelial cell differentiation.

Platelet-derived growth factor BB (PDGF-BB) has been assigned a critical role in vascular stability by promoting the recruitment of PDGF receptor-beta-expressing perivascular cells. Here we present data indicating that early hematopoietic/endothelial (hemangio) precursors express PDGFR-beta based on coexpression with CD31, vascular endothelial growth factor receptor-2, and CD41 in 2 models: mouse yolk sac (embryonic day 8 [E8]) and differentiating mouse embryonic stem cells (embryoid bodies). Expression of PDGFR-beta on hemangioprecursor cells in the embryoid bodies gradually disappeared, and, at E14, expression appeared on perivascular cells. Activation of the PDGFR-beta on the hemangioprecursors accelerated the differentiation of endothelial cells, whereas differentiation of the hematopoietic lineage was suppressed. In E9.5 yolk sacs derived from recombinant mice expressing kinase-active PDGFR-beta with an aspartic acid to asparagine (D894N) replacement in the kinase activating loop and from mice with ubiquitous expression of PDGF-BB driven by the Rosa26 locus, the number of CD41-expressing early hematopoietic cells decreased by 36% and 34%, respectively, compared with staged wild-type littermates. Moreover, enhanced vascular remodeling was evident in the Rosa26-PDGF-BB yolk sacs. We conclude that PDGFR-beta is expressed on early hemangioprecursor cells, regulating vascular/hematopoietic development.