Ciprian P. Gheorghe

Brain Renin-Angiotensin System: Fetal Epigenetic Programming by Maternal Protein Restriction During Pregnancy

Objective: Maternal protein malnutrition during pregnancy can lead to significant alterations in the systemic renin-angiotensin system (RAS) in the fetus. All components of the RAS are present in brain and may be altered in many disease states. Importantly, these disorders are reported to be of higher incidence in prenatally malnourished individuals. In the current study, we tested the hypothesis that antenatal maternal low protein diet (MLPD) leads to epigenetic changes and alterations in gene expression of brain RAS of the mouse fetus. Methods: Mice dams were given control and 50% MLPD during second half of the gestation. We analyzed messenger RNA (mRNA), microRNA (miRNA), promoter DNA methylation, and protein expression of various RAS genes in the fetal offspring. Results: As a consequence of 50% MLPD, fetal brains showed increased mRNA expression of angiotensinogen and angiotensin converting enzyme-1 (ACE-1), with a decrease in mRNA levels of angiotensin II type-2 (AT2) receptors. In contrast, while angiotensinogen protein expression was unaltered, the protein levels of ACE-1 and AT2 receptor genes were significantly reduced in the fetal brain from the MLPD dams. Our results also demonstrated hypomethylation of the CpG islands in the promoter regions of ACE-1 gene, and upregulation of the miRNAs, mmu-mir-27a and 27b, which regulate ACE-1 mRNA translation. Furthermore, our study showed reduced expression of the miRNA mmu-mir-330, which putatively regulates AT2 translation. Conclusion: For the developing fetal brain RAS, MLPD leads to significant alterations in the mRNA and protein expression, with changes in DNA methylation and miRNA, key regulators of hypertension in adults.

Gene expression in the placenta: maternal stress and epigenetic responses

Successful placental development is crucial for optimal growth, development, maturation and survival of the embryo/fetus into adulthood. Numerous epidemiologic and experimental studies have demonstrated the profound influence of intrauterine environment on life, and the diseases to which one is subject as an adult. For the most part, these invidious influences, whether maternal hypoxia, protein or caloric deficiency or excess, and others, represent types of maternal stress. In the present review, we examine certain aspects of gene expression in the placenta as a consequence of maternal stressors. To examine these issues in a controlled manner, and in a species in which the genome has been sequenced, most of these reported studies have been performed in the mouse. Although each individual maternal stress is characterized by up- or down-regulation of specific genes in the placenta, functional analysis reveals some patterns of gene expression common to the several forms of stress. Of critical importance, these genes include those involved in DNA methylation and histone modification, cell cycle regulation, and related global pathways of great relevance to epigenesis and the developmental origins of adult health and disease.

Placental Gene Expression Responses to Maternal Protein Restriction in the Mouse

OBJECTIVE Maternal protein restriction has been shown to have deleterious effects on placental development, and has long-term consequences for the progeny. We tested the hypothesis that, by the use of microarray technology, we could identify specific genes and cellular pathways in the developing placenta that are responsive to maternal protein deprivation, and propose a potential mechanism for observed gene expression changes. METHODS We fed pregnant FVB/NJ mice from day post-coitum 10.5 (DPC10.5) to DPC17.5, an isocaloric diet containing 50% less protein than normal chow. We used the Affymetrix Mouse 430A_2.0 array to measure gene expression changes in the placenta. We functionally annotated the regulated genes, and examined over-represented functional categories and performed pathway analysis. For selected genes, we confirmed the microarray results by use of qPCR. RESULTS We observed 244 probe sets, corresponding to 235 genes, regulated by protein restriction (p<0.001), with ninety-one genes being up-regulated, and 153 down-regulated. Up-regulated genes included those involved in the p53 pathway, apoptosis, negative regulators of cell growth, negative regulators of cell metabolism and genes related to epigenetic control. Down-regulated genes included those involved in nucleotide metabolism. CONCLUSIONS Microarray analysis has allowed us to describe the genetic response to maternal protein deprivation in the mouse placenta. We observed that negative regulators of cell growth and metabolism in conjunction with genes involved in epigenesis were up-regulated, suggesting that protein deprivation may contribute to growth restriction and long-term epigenetic changes in stressed tissues and organs. The challenge will be to understand the cellular and molecular mechanisms of these gene expression responses.

Gene Expression Patterns in the Hypoxic Murine Placenta: A Role in Epigenesis?

Hypoxia has been identified as a major stress or in placental and fetal development. To test the hypothesis that hypoxic stress responses are associated with gene expression changes, the authors measured gene expression in the mouse placenta in response to 48 hours of hypoxia. Embryonic day 15.5 pregnant mice were exposed to 48 hours of hypoxia (10.5% O2), after which the Affymetrix Mouse 430A_2.0 array was used to measure gene expression changes in the placenta. The authors observed 171 probe sets, corresponding to 163 genes, that were regulated by hypoxia (P < .01). Ninety genes were upregulated, and 73 were downregulated. The authors functionally annotated the regulated genes and examined overrepresented functional categories. Among the upregulated and downregulated genes, several overrepresented functional categories were observed. Upregulated genes included those involved in metabolism, oxygen transport, proteolysis, cell death, metabolism of reactive oxygen species, and DNA methylation. Genes involved in transcription, cell cycle regulation, and cell structure were downregulated. Microarray analysis has allowed the description of the genetic responses to hypoxia in the mouse placenta. The observation that hypoxia upregulates reactive oxygen species metabolism, in conjunction with DNA methylation enzymes, suggests that hypoxia may contribute to long-term epigenetic changes in stressed fetal tissues and organs.

Antenatal maternal hypoxic stress: adaptations in fetal lung Renin-Angiotensin system.

Antenatal maternal hypoxia (AMH) can lead to intrauterine growth restriction (IUGR), as well as idiopathic pulmonary hypertension of newborn and adult, the latter of which may be a consequence of alterations in the local pulmonary renin-angiotensin system (RAS). Little is known of these adaptations, however. Thus, we tested the hypothesis that antenatal maternal hypoxia is associated with alterations in gene and protein expression of the pulmonary renin-angiotensin system, which may play an important role in pulmonary disorders in the offspring. In FVB/NJ mice, we studied messenger RNA (mRNA) and protein expression, as well as promoter DNA methylation and microRNA (miRNA) levels in response to 48 hours hypoxia (10.5% O2) at 15.5 day post coitum (DPC). In response to AMH, the pulmonary mRNA levels of angiotensin-converting enzyme (ACE) 1.2, ACE-2, and angiotensin II type 1b (AT-1b) receptors were increased significantly, as compared to controls (N = 4). In response to antenatal hypoxia, pulmonary protein levels of renin and ACE-2 also were increased significantly, whereas ACE-1 protein expression was reduced. In fetal lungs, we also observed reduced expression of the miRNAs: mmu-mir −199b, −27b, −200b, and −468 that putatively increase the translation of renin, ACE-1, ACE-2, and AT-1 receptors, respectively. In response to AMH, promoter methylation of ACE was unchanged. We conclude that AMH leads to changes in expression of pulmonary RAS of fetal mice. The possible implications of these changes for the regulation of pulmonary vascular contractility in later life remain to be explored.

Maternal Protein Deprivation: Changes in Systemic Renin-Angiotensin System of the Mouse Fetus

We tested the hypothesis that maternal protein deprivation during gestation results in changes in expression of the systemic renin-angiotensin system in fetal mice. Fetal weight was decreased significantly as a consequence of 50% maternal protein deprivation during second half of gestation. In fetal liver, angiotensinogen protein expression was reduced significantly despite a significant increase in messenger RNA (mRNA). In fetal kidneys, both mRNA and protein levels of renin were increased significantly. In the lungs, we observed a decrease in both angiotensin-converting enzyme I and II mRNA expression, whereas protein expression of both isoforms was increased significantly. The fetal heart showed significant increases in expression of angiotensin II type 1 (AT-1) and type 2 (AT-2) receptors mRNA. Protein expression of AT-1 receptors increased, while that of AT-2 receptors decreased. We conclude that maternal low-protein diet during gestation leads to significant changes in expression of the systemic renin-angiotensin system in fetal mice and may be important in the genesis of hypertension in the adult.

Gene expression patterns in the developing murine placenta.

Objective: Successful placental development is crucial for optimal growth, maturation, and survival of the embryo/fetus. To examine genetic aspects of placental development, use investigated gene expression patterns in the murine placenta at embryonic day 10.5 (E10.5), E12.5, E15.5, and E17.5. Methods: By use of the Affymetrix MU74A array (Affymetrix, Santa Clara, CA), we measured expression levels for 12,473 probe sets. Using pairwise analysis we selected 622 probe sets, corresponding to 599 genes, that were up- or down-regulated by more than fourfold between time points E10.5 and E12.5, E12.5 and E15.5, E15.5 and E17.5. We analyzed and functionally annotated those genes regulated during development. Results: In comparing E10.5 to E12.5 we found that angiogenesis and fatty acid metabolism and transport related genes were up-regulated at E10.5, while genes involved in hormonal control and ribosomal proteins were up-regulated at E12.5. When comparing E12.5 to E15.5 we noted that genes involved in the cell cycle and RNA metabolism were strongly up-regulated at E12.5, while genes involved in cellular transport were up-regulated at E15.5. Finally, when comparing E15.5 to E17.5, we found genes related to cell cycle control, genes expressed in the nucleus and involved in RNA metabolism were up-regulated at E17.5. Conclusion: Microarray analysis has allowed us to describe gene expression patterns and profils in the developing mouse placenta. Further analysis has demonstrated that several functional classes are up- and down-regulated at specific time points in placental development. These changes may have significant implications for placental development in the human.