M. A. Hale

Fetal growth restriction alters transcription factor binding and epigenetic mechanisms of renal 11β-hydroxysteroid dehydrogenase type 2 in a sex-specific manner

Intrauterine growth restriction (IUGR) increases the risk of serious adult morbidities such as hypertension. In an IUGR rat model of hypertension, we reported a persistent decrease in kidney 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) mRNA and protein levels from birth through postnatal (P) day 21. This enzyme deficiency can lead to hypertension by limiting renal glucocorticoid deactivation. In the present study, we hypothesized that IUGR affects renal 11beta-HSD2 epigenetic determinants of chromatin structure and alters key transcription factor binding to the 11beta-HSD2 promoter in association with persistent downregulation of its mRNA expression. To test this hypothesis, we performed bilateral uterine artery ligation on embryonic day 19.5 pregnant rats and harvested kidneys at day 0 (P0) and P21. Key transcription factors that can affect 11beta-HSD2 expression include transcriptional enhancers specificity protein 1 (SP1) and NF-kappaB p65 and transcriptional repressors early growth response factor (Egr-1) and NF-kappaB p50. Our most important findings were as follows: 1) IUGR significantly decreased SP1 and NF-kappaB (p65) binding to the 11beta-HSD2 promoter in males, while it increased Egr-1 binding in females and NF-kappaB (p50) binding in males; 2) IUGR increased CpG methylation status, as well as modified the pattern of methylation in several CpG sites of 11beta-HSD2 promoter at P0 also in a sex-specific manner; and 3) IUGR decreased trimethylation of H3K36 in exon 5 of 11beta-HSD2 at P0 and P21 in both genders. We conclude that IUGR is associated with altered transcriptional repressor/activator binding in connection with increased methylation in the 11beta-HSD2 promoter region in a sex-specific manner, possibly leading to decreased transcriptional activity. Furthermore, IUGR decreased trimethylation of H3K36 of the 11beta-HSD2 gene in both genders, which is associated with decreased transcriptional elongation. We speculate that alterations in transcription factor binding and chromatin structure play a role in in utero reprogramming.

Uteroplacental insufficiency affects kidney VEGF expression in a model of IUGR with compensatory glomerular hypertrophy and hypertension

Low nephron endowment secondary to intrauterine growth restriction (IUGR) results in compensatory hypertrophy of the remaining glomeruli, which in turn is associated with hypertension. However, gender differences exist in the response of the kidney to injury, and IUGR female offspring seems protected from an unfavorable outcome. We previously reported differences in gender-specific gene expression in the IUGR kidney as well as increased circulating corticosterone levels following uteroplacental insufficiency (UPI). Vascular endothelial growth factor (VEGF), which is critical for renal development, is an important candidate in the IUGR kidney since its expression can be regulated by sex-steroids and glucocorticoids. We hypothesize that IUGR leads to altered kidney VEGF expression in a gender-specific manner. Following uterine ligation in the pregnant rat, UPI decreases renal VEGF levels in male and female IUGR animals at birth and through postnatal day 21. However, by day 120 of life, IUGR females have increased kidney VEGF expression, not present in the IUGR males. In addition, IUGR males exhibit increased serum testosterone levels as well as proteinuria. These findings are intriguing in light of the difference in glomerular hypertrophy observed: IUGR males show increased glomerular area when compared to IUGR females. In this model characterized by decreased nephron number and adult onset hypertension, UPI decreases renal VEGF expression during nephrogenesis. Our most intriguing finding is the increased renal VEGF levels in adult IUGR females, associated with a more benign phenotype. We suggest that the mechanisms underlying renal disease in response to IUGR are most likely regulated in a gender specific manner.

Intrauterine Growth Restriction Alters T-Lymphocyte Cell Number and Dual Specificity Phosphatase 1 Levels in the Thymus of Newborn and Juvenile Rats

Intrauterine growth restricted (IUGR) infants have increased susceptibility to infection associated with higher risk of illness and death. Dual specificity phosphatase 1 (DUSP1), which is transcribed in the thymus, increases in quantity as T cells mature and differentiate into CD4+ cells. Little is known about how IUGR affects DUSP1 levels and T-cell subpopulations over time. We hypothesized that IUGR would decrease cell count, CD4+ and CD8+ subpopulations of T lymphocytes, and DUSP1 levels in IUGR rat thymus and spleen. Bilateral uterine artery ligation produced IUGR rats. Thymus and spleen were harvested at P0 and P21. Flow cytometry was used to compare CD4+ and CD8+ lymphocyte populations. Real-time RT-PCR and Western blotting were used to determine DUSP1 quantity. IUGR significantly decreased total cell count in P0 and P21 IUGR male and female thymus. IUGR significantly increased CD4+ cells in IUGR P0 males and females, significantly decreased CD4+ cells in P21 female thymus, and significantly altered DUSP1 levels in the IUGR female thymus at P0 and P21, although it is not yet known whether the change in DUSP1 levels is due to a change in the level per cell or to a change in cellular composition of the thymus.

Intrauterine growth restriction transiently delays alveolar formation and disrupts retinoic acid receptor expression in the lung of female rat pups

Background:We showed that intrauterine growth restriction (IUGR) increases distal airspace wall thickness at birth (postnatal age 0; P0) in rat pups (saccular stage of lung development). However, that report did not assess whether the saccular phenotype persisted postnatally or occurred in males or females, nor did the report identify a potential molecular pathway for the saccular phenotype at P0. We hypothesized that IUGR persistently delays alveolar formation and disrupts retinoic acid receptor (RAR) mRNA and protein levels in the lung of rat pups in a postnatal age– and sex-specific manner.Methods:IUGR was induced in pregnant rats by bilateral uterine artery ligation. Alveolar formation and expression of RARα, -β, and -γ were quantified at P0, P6 (alveolar stage), and P21 (postalveolarization).Results:IUGR increased distal airspace wall thickness in female pups at P0 only. IUGR did not affect male pups at any age. IUGR transiently increased lung RAR-β protein abundance, which inhibits alveolar formation, at P0 in female pups. Serum retinol concentration was normal at all ages.Conclusion:IUGR alone is not sufficient to persistently delay postnatal alveolar formation or disrupt expression of RARs. We speculate that for IUGR to delay alveolar formation postnatally, a second insult is necessary.

Altered expression and chromatin structure of the hippocampal IGF1r gene is associated with impaired hippocampal function in the adult IUGR male rat.

Exposure to intrauterine growth restriction (IUGR) is an important risk factor for impaired learning and memory, particularly in males. Although the basis of IUGR-associated learning and memory dysfunction is unknown, potential molecular participants may be insulin-like growth factor 1 (Igf1) and its receptor, IGF1r. We hypothesized that transcript levels and protein abundance of Igf1 and IGF1r in the hippocampus, a brain region critical for learning and memory, would be lower in IUGR male rats than in age-matched male controls at birth (postnatal day 0, P0), at weaning (P21) and adulthood (P120). We also hypothesized that changes in messenger Ribonucleic acid (mRNA) transcript levels and protein abundance would be associated with specific histone marks in IUGR male rats. Lastly, we hypothesized that IUGR male rats would perform poorer on tests of hippocampal function at P120. IUGR was induced by bilateral ligation of the uterine arteries in pregnant dams at embryonic day 19 (term is 21 days). Hippocampal Igf1 mRNA transcript levels and protein abundance were unchanged in IUGR male rats at P0, P21 or P120. At P0 and P120, IGF1r expression was increased in IUGR male rats. At P21, IGF1r expression was decreased in IUGR male rats. Increased IGF1r expression was associated with more histone 3 lysine 4 dimethylation (H3K4Me2) in the promoter region. In addition, IUGR male rats performed poorer on intermediate-term spatial working memory testing at P120. We speculate that altered IGF1r expression in the hippocampus of IUGR male rats may play a role in learning and memory dysfunction later in life.

Isoflurane Exposure did not Adversely Affect Recognition Memory or Decrease Hippocampal Brain Derived Neurotrophic Factor Expression in the 17 Day Old Rat Pup

A previous study showed that hippocampal BDNF mRNA decreased in 17 day old (D17) rats, relative to agematched naive rats, at day 1, 3, 7 and 14 after sham surgery in a traumatic brain injury model. The anesthetic isoflurane activates GABA and inhibits NMDA receptor currents, both of which are known to decrease Brain-Derived Neurotrophic Factor (BDNF) mRNA in rat hippocampi. Hippocampal BDNF is necessary for normal cognitive function. Effects of isoflurane alone on hippocampal BDNF are not known. We hypothesized that, in D17 rat pups, isoflurane would decrease hippocampal mRNA/protein levels of BDNF and Synapsin I (a downstream target of BDNF important for cognitive function) and impair performance on the Novel Object Recognition Test (NOR). ISOF BDNF and Synapsin I mRNA decreased relative to Naive at day 1 and 8 after exposure, but not at day 14. Isoflurane exposure did not decrease hippocampal protein levels of BDNF or Synapsin I and did not impair NOR performance. In contrast to the neonatal rat pup, anesthetic exposure did not impair cognitive function. We speculate that adverse effects of anesthetics on rat pup cognitive function and BDNF expression are highly dependent on age at exposure.

306 INTRAUTERINE GROWTH RESTRICTION DECREASES IMMATURE T CELLS IN THE THYMUS OF JUVENILE RATS.

Background IUGR infants have qualitative and quantitative abnormalities in lymphocyte function that persist into childhood, resulting in increased susceptibility to infections. We have previously shown in a rat model of IUGR that there is a decrease in the overall number of lymphocytes. However, little detail is known about the extent to which IUGR affects all white blood cells. Interestingly, lymphocyte phenotype can be altered by high glucocorticoid levels, which also characterizes both IUGR human and rat. Objective We hypothesized that IUGR affects leukocyte phenotype in postnatal juvenile IUGR rats. Design/Methods To prove this hypothesis, we used bilateral uterine artery ligation and sham surgery to produce IUGR and control rats (n = 4 in each group). Perinatal glucocorticoid levels are elevated in this model of IUGR (Baserga et al, AJP, 2005). Pups were allowed to deliver, and litters were culled to 6. Pups were killed on day of life 21, and thymus and spleen were harvested. Organ weight and total cell number were corrected for body weight differences. Monoclonal antibodies and flow cytometry analysis were used to compare the major leukocyte cell lines in the thymus and spleen from 21-day-old juvenile rats. Cells were stained with cell-surface markers for general T cells (CD3+), T-helper (CD4+), T-cytotoxic (CD8+), granulocytes (GR1+), B cells (CD45r+), macrophages (Mac1+), and dendritic cells (CD11c+). Results Thymus and spleen weights were disproportionately decreased in IUGR. IUGR decreased double positive (CD4+, CD8+) naïve, undifferentiated T cells in the thymus versus controls (72 ± 1% vs 66 ± 1%, p < .05). Additionally, IUGR increased the percentage of more mature, cytotoxic T cells, dendritic and B cells in the thymus. Similarly, the IUGR spleen had a significant shift toward a more mature and cytotoxic phenotype. Conclusions We conclude that IUGR disproportionately decreases the naïve, undifferentiated T cells in the postnatal thymus and shifts the percentage of cells to a more mature, differentiated phenotype. This is very intriguing because increased glucocorticoid levels have been previously shown to similarly affect lymphocyte phenotype. We speculate that elevated perinatal glucocorticoid levels lead to a persistent change in the postnatal lymphocyte population in the IUGR rat, which represents a pathway through which IUGR causes qualitative and quantitative abnormities in lymphocyte function. Supported by the Childrens Health Research Center.

443 INTRAUTERINE GROWTH RETARDATION AFFECTS POSTNATAL RIBONUCLEIC ACID LEVELS OF PROTEINS INVOLVED IN RETINOIC ACID SIGNALING AND METABOLISM.

Introduction Intrauterine growth retardation (IUGR) affects postnatal hepatic gene expression. Many of these changes are secondary to epigenetic modifications of chromatin, which are often gender specific. The gender-specific effects of IUGR upon chromatin structure widens from day of life (DOL) 0 through DOL 21. The signaling mechanism responsible for the progressive difference between the genders is unknown. Preliminary data from differential display RT-PCR experiments suggest that IUGR affects mRNA levels of protein involved in retinoic acid metabolism. These findings are intriguing because retinoic acid and its receptors are known to alter chromatin structure in multiple cell culture systems. Hypothesis We hypothesized that IUGR would alter mRNA levels of the following genes involved in retinoic acid signaling and metabolism: RARα (receptor), RARβ (receptor), RARγ (receptor), aldh1a1 (synthesis), aldh1a2 (synthesis), CYP26a1 (degradation), and Rbp1 (transport). Methods Induction of IUGR was done through bilateral uterine artery ligation of the pregnant rat at day 19 of gestation with delivery by cesarean section on day 21.5 (term) of gestation. Using liver tissue from day 0 and day 21 rat pups, mRNA was isolated, cDNA synthesized, and RT-PCR used to analyze the genes listed above. Results IUGR significantly decreased RARγ expression in DOL21 female livers (p Conclusion IUGR induced by uteroplacental insufficiency increases mRNA levels of RARγ in female IUGR liver and decreases mRNA levels of CYP26a1 in male IUGR livers. These gender-specific differences are intriguing, considering that they parallel the gender-specific differences in chromatin structure and histone modifications previously described. Furthermore, the decrease in male CYP26a1 levels suggests that the male IUGR rat is vitamin A deficient, despite 3 weeks of ad libitum feeding. We speculate that the gender-specific differences in RA signaling and metabolism may play a role in the progressive gender differences observed in the IUGR rat.

UTEROPLACENTAL INSUFFICIENCY AFFECTS RENAL EXPRESSION OF ESTROGEN RECEPTORS IN INTRAUTERINE GROWTH-RESTRICTED RATS.: 65

Background Uteroplacental insufficiency results in intrauterine growth restriction (IUGR). IUGR newborns are at an increased risk for later development of morbidities, including hypertension, at an incidence that varies between genders. Although we have previously demonstrated differences in gender-specific gene expression in the IUGR rat kidney, it is unknown whether IUGR affects kidney expression of either the androgen or estrogen receptors. Objective We hypothesized that IUGR would alter renal mRNA levels of the androgen receptor (AR), estrogen receptor 1 (ER1), and estrogen receptor 2 (ER2) in a gender-specific manner in day 0 (d0) and day 21 (d21) IUGR rat pups. Methods Bilateral uterine artery ligation was performed on day 19 pregnant Sprague-Dawley rats to render them IUGR, and pups were then harvested on day 21 (term). Levels of AR, ER1, and ER2 mRNA were quantified using real-time RT-PCR from whole kidneys on days 0 and 21. Results mRNA levels expressed as % of control ⊆ SEM. IUGR significantly increased renal ER1 mRNA levels in d21 males to 177 ⊆ 16.* In contrast, ER2 mRNA levels were significantly decreased to 57 ⊆ 6** in day 21 female kidneys. Identical changes in the ER1 and ER2 mRNA levels were seen in day 0 males and females but did not achieve statistical significance. *p Conclusions IUGR increases ER1 receptor expression in IUGR male rats while decreasing ER2 receptor expression in IUGR female rats. Interestingly, the differences in gene expression widens from day 0 to day 21 in the IUGR rat kidney. We speculate that such differences contribute to the gender-specific characteristics of the IUGR rat kidney.

UTEROPLACENTAL INSUFFICIENCY CAUSES CHANGES IN LEPTIN RECEPTOR EXPRESSION IN THE INTRAUTERINE GROWTH RETARDED RAT BRAIN.: 442

Background Uteroplacental insufficiency causes intrauterine growth retardation (IUGR). IUGR has been linked to a variety of metabolic and developmental disorders, including diabetes, obesity, infertility, stunted skeletal growth, and impaired brain growth. Such disorders may be mediated through the direct affect of IUGR on cerebral proteins and receptors involved in reproductive and metabolic pathways. Leptin and ghrelin have been linked to energy homeostasis, reproductive development, and neuronal synthesis, but the expression of leptin and ghrelin receptors (Ob-Rb and GHS-R, respectively) in the whole brain of IUGR rat pups is unknown. Additionally, the effect of IUGR on kisspeptin (KiSS-1), a protein regulated by leptin and recently implicated in development of the reproductive axis, is unknown. Objectives We hypothesized that uteroplacental insufficiency would cause increased expression of Ob-Rb and GHS-R and decreased expression of KiSS-1 in the whole brain of IUGR rats. Methods To test this, we measured expression of Ob-Rb, GHS-R, and KiSS-1 in the whole brain of IUGR pups obtained by bilateral uterine artery ligation. Sham surgeries were used as controls. Brains were harvested and flash frozen. Real-time RT-PCR was used to measure mRNA expression on day 0 (D0) and day 21 (D21) of life. Results Although there was no significant difference between IUGR and SHAM rats in GHS-R or KiSS-1 expression, D0 IUGR pups had significantly higher expression of Ob-Rb mRNA in whole brain than D0 SHAM pups (p = .003). Preliminary results indicate a trend toward decreased expression of Ob-Rb mRNA in whole brain of D21 IUGR rats (p = .09). Conclusion We conclude that uteroplacental insufficiency up-regulates Ob-Rb expression at D0 in the IUGR rat brain. We have shown that the IUGR insult epigenetically regulates cerebral gene expression, and we speculate that Ob-Rb expression may be modulated through changes in chromatin structure. Additionally, recent research has demonstrated that leptin signaling affects adrenal glucocorticoid production as well as CNS glucocorticoid receptor expression. We have previously shown that IUGR affects glucocorticoid receptor expression in the rat brain, and we postulate that this effect may be mediated through up-regulation of cerebral Ob-Rb receptors.