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.

SOD1 deficiency causes salt sensitivity and aggravates hypertension in hydronephrosis.

Hydronephrosis causes renal dysfunction and salt-sensitive hypertension, which is associated with nitric oxide deficiency and abnormal tubuloglomerular feedback (TGF) response. We investigated the role of oxidative stress for salt sensitivity and for hypertension in hydronephrosis. Hydronephrosis was induced in superoxide dismutase 1-transgenic (SOD1-tg), SOD1-deficient (SOD1-ko), and wild-type mice and in rats. In mice, telemetric measurements were performed during normal (0.7% NaCl) and high-sodium (4% NaCl) diets and with chronic tempol supplementation. The 8-iso-prostaglandin-F(2alpha) (F2-IsoPs) and protein excretion profiles and renal histology were investigated. The acute effects of tempol on blood pressure and TGF were studied in rats. In hydronephrosis, wild-type mice developed salt-sensitive hypertension (114 +/- 1 to 120 +/- 2 mmHg), which was augmented in SOD1-ko (125 +/- 3 to 135 +/- 4 mmHg) but abolished in SOD1-tg (109 +/- 3 to 108 +/- 3 mmHg). SOD1-ko controls displayed salt-sensitive blood pressure (108 +/- 1 to 115 +/- 2 mmHg), which was not found in wild types or SOD1-tg. Chronic tempol treatment reduced blood pressure in SOD1-ko controls (-7 mmHg) and in hydronephrotic wild-type (-8 mmHg) and SOD1-ko mice (-16 mmHg), but had no effect on blood pressure in wild-type or SOD1-tg controls. SOD1-ko controls and hydronephrotic wild-type and SOD1-ko mice exhibited increased fluid excretion associated with increased F2-IsoPs and protein excretion. The renal histopathological changes found in hydronephrotic wild-type were augmented in SOD1-ko and diminished in SOD-tg mice. Tempol attenuated blood pressure and normalized TGF response in hydronephrosis [DeltaP(SF): 15.2 +/- 1.2 to 9.1 +/- 0.6 mmHg, turning point: 14.3 +/- 0.8 to 19.7 +/- 1.4 nl/min]. Oxidative stress due to SOD1 deficiency causes salt sensitivity and plays a pivotal role for the development of hypertension in hydronephrosis. Increased superoxide formation may enhance TGF response and thereby contribute to hypertension.

Evidence of increased oxidative stress and a change in the plasminogen activator inhibitor (PAI)-1 to PAI-2 ratio in early-onset but not late-onset preeclampsia.

OBJECTIVE: The aims of this study were to measure the degree of oxidative stress and alterations in plasminogen activator inhibitor (PAI) type 1 and PAI-2 ratio in women with early-onset and late-onset preeclampsia. STUDY DESIGN: A case-control study was conducted in women with early-onset (24-32 weeks' gestation; n=18) and late-onset (35-42 weeks' gestation; n=20) preeclampsia and in control pregnant women at corresponding gestational weeks. Placenta, urine, and serum samples were collected. RESULTS: In early-onset preeclampsia, the median placental concentration of 8-iso-prostaglandin (PG)-F2alpha was higher and the PAI-1 to PAI-2 ratio higher than in early controls. These values did not differ between women with late-onset preeclampsia and their corresponding controls. Serum concentrations of 8-iso-PGF2alpha and vitamins C and E did not differ between cases and controls. CONCLUSION: Early-onset but not late-onset preeclampsia is associated with increased placental oxidative stress and increased PAI-1 to PAI-2 ratio.

Congenital anomalies in diabetic pregnancy.

Congenital malformations are more common in infants of diabetic women than in children of non-diabetic women. The etiology, pathogenesis and prevention of the diabetes-induced malformations have spurred considerable clinical and basic research efforts. The ultimate aim of these studies has been to obtain an understanding of the teratogenic process, which may enable precise preventive therapeutic measures in diabetic pregnancies. The results of the clinical and basic studies support the view of an early gestational induction of the malformations in diabetic pregnancy by a teratogenic process of multifactorial etiology. There may be possible targets for new therapeutic efforts revealed by the research work. Thus, future additions to the therapeutic efforts may include supplementation with antioxidants and/or folic acid, although more research is needed to delineate the dosages and compounds to be used. As the research into genetic predisposition for the teratogenic induction of malformations by maternal diabetes starts to reveal new genes and gene products involved in the etiology of the malformations, a set of new targets for intervention may arise.

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.

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.

Early postpartum changes in circulating pro- and anti-angiogenic factors in early-onset and late-onset pre-eclampsia.

BACKGROUND: Pre-eclampsia is associated with altered plasma concentrations of the pro- and anti-angiogenic factors placental growth factor (PlGF), vascular endothelial growth factor-A (VEGF-A) and soluble fms-like tyrosine kinase 1 (sFlt1). However, there is insufficient knowledge about how these concentrations change after delivery, and whether the changes differ between early-onset and late-onset pre-eclampsia. METHODS: Plasma concentrations of sFlt1, PlGF and VEGF-A were measured before delivery and on days 1, 3 and 7 postpartum in women with early-onset (24-32 weeks' gestation) (n=18) and late-onset pre-eclampsia (35-42 weeks' gestation) (n=20) and in control groups delivered in corresponding weeks of gestation. RESULTS: All groups showed a rapid decrease in median sFlt1 concentration postpartum. Women with late-onset pre-eclampsia did not differ in sFlt1 concentration from controls on day 1 postpartum, whereas women with early-onset pre-eclampsia displayed a persistently elevated sFlt1 concentration on day 7 postpartum compared with controls. PlGF concentrations did not change from before delivery to any time point postpartum in the pre-eclampsia groups. VEGF-A concentrations were slightly increased on day 7 postpartum in both pre-eclampsia and control groups compared to concentrations prior to delivery. CONCLUSION: Median sFlt1 concentrations decreased rapidly postpartum in all groups, but remained higher in early-onset pre-eclampsia than controls on day 7. Postpartum, the median PlGF concentrations were similar to the concentrations measured before delivery in women with pre-eclampsia.

Placental growth factor and soluble FMS-like tyrosine kinase-1 in early-onset and late-onset preeclampsia.

OBJECTIVE: To estimate whether alterations in plasma levels of the proangiogenic proteins placental growth factor (PlGF) and vascular endothelial growth factor-A (VEGF-A), and the antiangiogenic protein soluble fms-like tyrosine kinase-1 (sFlt1) were more pronounced in early-onset than in late-onset preeclampsia. METHODS: A cross-sectional study was conducted to estimate the levels of sFlt1, PlGF, and VEGF-A in plasma in a control group of nonpregnant women, in an early control group of women at 24-32 weeks of gestation, in a late control group of women at 36-42 weeks of gestation, and in cases of women with early-onset (before 32 weeks of gestation) and late-onset (after 35 weeks of gestation) preeclampsia. RESULTS: Women with early-onset preeclampsia had a 43 times higher median plasma sFlt1 level than early controls (P<.001). Women with late-onset preeclampsia had a three times higher median plasma sFlt1 level than late controls (P<.001). Women with early-onset preeclampsia had a 21 times lower median plasma PlGF level than early controls (P<.001). Women with late-onset preeclampsia had a five times lower median plasma PlGF level than late controls (P=.01). The median level of VEGF-A in plasma was less than 15 pg/mL in all groups of pregnant women. CONCLUSION: Both early- and late-onset preeclampsia are associated with altered plasma levels of sFlt1 and PlGF. The alterations are more pronounced in early-onset rather than in late-onset disease.

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.

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.

Folic acid supplementation diminishes diabetes- and glucose-induced dysmorphogenesis in rat embryos in vivo and in vitro.

Maternal administration of folic acid diminishes the risk of neural tube defects (NTDs) in offspring, but whether folic acid exerts a similar effect in diabetic pregnancy is unknown. The aim was to investigate whether maldevelopment in rat embryos caused by exposure to diabetes in vivo or high-glucose concentrations in vitro is affected by subcutaneous administration of folic acid to the pregnant mother or by adding the compound to the culture medium, respectively. Exposure of embryos to maternal diabetes in vivo or 30 mmol/l glucose in vitro yielded an increased malformation rate (71 and 88% NTD, respectively) and lowered somite number and crown-rump length compared with control embryos. When we injected folic acid into the diabetic pregnant rat, or added 2 mmol/l folic acid to the culture medium with high glucose, the embryonic parameters improved (3 and 5% NTD, respectively). The present work shows that administration of folic acid can diminish diabetes-induced maldevelopment. This suggests that folic acid supplementation may have a role in the prevention of malformations in diabetic pregnancy.

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.

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.

Receptor for advanced glycation end products (RAGE) knockout reduces fetal dysmorphogenesis in murine diabetic pregnancy.

The receptor for Advanced Glycation End products (RAGE) is implicated in the pathogenesis of diabetic complications, but its importance in diabetic embryopathy is unclear. We therefore investigated the role of RAGE in diabetic embryopathy using streptozotocin induced diabetes in female wild type (WT) C57Bl/6N and RAGE knockout C57Bl/6N (RAGE(-/-)) mice, mated with control males of the same genotype. Maternal diabetes induced more fetal resorption and malformation (facial skeleton, neural tube) in the WT than in the RAGE(-/-) fetuses. Maternal plasma glucose and methylgyoxal concentrations, as well as embryonic N(ε)-carboxymethyl-lysine (CML) levels were increased to the same extent in diabetic WT and RAGE(-/-) pregnancy. However, maternal diabetes induced increased fetal hepatic isoprostane 8-iso-PGF2α levels (oxidative stress marker) and embryonic activation of NFκB in WT only (not in RAGE(-/-) embryos). The association between RAGE knockout and diminished embryonic dysmorphogenesis in diabetic pregnancy suggests that embryonic RAGE activation is involved in diabetic embryopathy.

Maternal diabetes in vivo and high glucose in vitro diminish GAPDH activity in rat embryos.

The aim of the present study was to investigate whether diabetic embryopathy may be associated with the inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) resulting from an excess of reactive oxygen species (ROS) in the embryo. Recent demonstrations of enhanced ROS production in mitochondria of bovine aortic endothelial cells exposed to high glucose have supported the idea that the pathogenesis of diabetic complications may involve ROS-induced GAPDH inhibition. We investigated whether a teratogenic diabetic environment also inhibits embryonic GAPDH activity and alters GAPDH gene expression and whether antioxidants diminish such GAPDH inhibition. In addition, we determined whether the inhibition of GAPDH with iodoacetate induces dysmorphogenesis, analogous to that caused by high glucose concentration, and whether antioxidants modulated the putative teratogenic effect of such direct GAPDH inhibition. We found that embryos from diabetic rats and embryos cultured in high glucose concentrations showed decreased activity of GAPDH (by 40-60%) and severe dysmorphogenesis on gestational days 10.5 and 11.5. GAPDH mRNA was decreased in embryos of diabetic rats compared to control embryos. Supplementing the high-glucose culture with the antioxidant N-acetylcysteine (NAC) increased GAPDH activity and diminished embryonic dysmorphogenesis. Embryos cultured with iodoacetate showed both decreased GAPDH activity and dysmorphogenesis; supplementing the culture with NAC increased both parameters toward normal values. In conclusion, dysmorphogenesis caused by maternal diabetes is correlated with ROS-induced inhibition of GAPDH in embryos, which could indicate that inhibition of GAPDH plays a causal role in diabetic embryopathy.

Placental dysfunction in Suramin-treated rats: impact of maternal diabetes and effects of antioxidative treatment.

OBJECTIVE: The aim of the present study was to evaluate a rat model of placental dysfunction/preeclampsia in pregnancies complicated by maternal diabetes. A second objective was to evaluate the effects of vitamin E treatment in this model. METHODS: Normal and streptozotocin-induced diabetic rats of two different strains (U and H) were given intraperitoneal (IP) injections of the angiogenesis inhibitor Suramin (Sigma Chemical Co, St Louis, MO) or saline in early pregnancy, and fed standard or vitamin E-enriched food. The outcome of pregnancy was evaluated on gestational day 20. RESULTS: In both rat strains Suramin caused fetal growth retardation, decreased placental blood flow, and increased placental concentration of the isoprostane 8-iso-PGF(2alpha). In the U rats Suramin also caused increased fetal resorption rate, increased maternal blood pressure, decreased renal blood flow, and diminished maternal growth. Diabetes caused severe maternal and fetal growth retardation, increased resorption rate, and increased placental 8-iso-PGF(2alpha) concentration independent of Suramin administration. The maternal and fetal effects of Suramin and diabetes were more pronounced in the U strain than in the H strain. Vitamin E treatment improved the status of Suramin-injected diabetic rats: in U rats the blood pressure increase was normalized; and in both U and H rats the decreased placental blood flow was marginally enhanced, and the increase in placental 8-iso-PGF(2alpha) was partly normalized by vitamin E. CONCLUSION: Suramin injections to pregnant rats cause a state of placental insufficiency, which in U rats resembles human preeclampsia. The induction of this condition is at least partly mediated by oxidative stress, and antagonized by antioxidative treatment. Maternal diabetes involves increased oxidative stress, and causes both maternal and fetal morbidity, which are only marginally affected by additional Suramin treatment.

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.

Linkage study of embryopathy-polygenic inheritance of diabetes-induced skeletal malformations in the rat.

We developed an inbred rat model of diabetic embryopathy, in which the offspring displays skeletal malformations (agnathia or micrognathia) when the mother is diabetic, and no malformations when she is not diabetic. Our aim was to find genes controlling the embryonic maldevelopment in a diabetic environment. We contrasted the fetal outcome in inbred Sprague-Dawley L rats (20% skeletal malformations in diabetic pregnancy) with that of inbred Wistar Furth rats (denotedW, no skeletal malformations in diabetic pregnancy). We used offspring from the backcross F(1)×L to probe for the genetic basis for malformation of the mandible in diabetic pregnancy. A set of 186 fetuses (93 affected, 93 unaffected) was subjected to a whole genome scan with 160 micro satellites. Analysis of genotype distribution indicated 7 loci on chromosome 4, 10 (3 loci), 14, 18, and 19 in the teratogenic process (and 14 other loci on 12 chromosomes with less strong association to the malformations), several of which contained genes implicated in other experimental studies of diabetic embryopathy. These candidate genes will be scrutinized in further experimentation. We conclude that the genetic involvement in rodent diabetic embryopathy is polygenic and predisposing for congenital malformations.

Superoxide dismutase 1 limits renal microvascular remodeling and attenuates arteriole and blood pressure responses to angiotensin II via modulation of nitric oxide bioavailability.

Oxidative stress is associated with vascular remodeling and increased preglomerular resistance that are both implicated in the pathogenesis of renal and cardiovascular disease. Angiotensin II induces superoxide production, which is metabolized by superoxide dismutase (SOD) or scavenged by NO. We investigated the hypothesis that SOD1 regulates renal microvascular remodeling, blood pressure, and arteriolar responsiveness and sensitivity to angiotensin II using SOD1-transgenic (SOD1-tg) and SOD1-knockout (SOD1-ko) mice. Blood pressure, measured telemetrically, rose more abruptly during prolonged angiotensin II infusion in SOD1-ko mice. The afferent arteriole media:lumen ratios were reduced in SOD1-tg and increased in SOD1-ko mice. Afferent arterioles from nontreated wild types had graded contraction to angiotensin II (sensitivity: 10(-9) mol/L; responsiveness: 40%). Angiotensin II contractions were less sensitive (10(-8) mol/L) and responsive (14%) in SOD1-tg but more sensitive (10(-13) mol/L) and responsive (89%) in SOD1-ko mice. Arterioles from SOD1-ko had 4-fold increased superoxide formation with angiotensin II at 10(-9) mol/L. N(G)-nitro-l-arginine methyl ester reduced arteriole diameter of SOD1-tg and enhanced angiotensin II sensitivity and responsiveness of wild-type and SOD1-tg mice to the level of SOD1-ko mice. SOD mimetic treatment with Tempol increased arteriole diameter and normalized the enhanced sensitivity and responsiveness to angiotensin II of SOD1-ko mice but did not affect wild-type or SOD1-tg mice. Neither SOD1 deficiency nor overexpression was associated with changes in nitrate/nitrite excretion or renal mRNA expression of NO synthase, NADPH oxidase, or SOD2/SOD3 isoforms and angiotensin II receptors. In conclusion, SOD1 limits afferent arteriole remodeling and reduces sensitivity and responsiveness to angiotensin II by reducing superoxide and maintaining NO bioavailability. This may prevent an early and exaggerated blood pressure response to angiotensin II.

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.