Intrauterine growth restriction (IUGR) and preeclampsia (PE) are placental pathologies known to complicate pregnancy and cause neonatal disorders. To date, there is a limited number of studies on the genetic similarity of these conditions. DNA methylation is a heritable epigenetic process that can regulate placental development. Our objective was to identify methylation patterns in placental DNA from normal, PE and IUGR-affected pregnancies. DNA was extracted, and bisulfite was converted, prior to being hybridized for the methylation array. Methylation data were SWAN normalized and differently methylated regions were identified using applications within the USEQ program. UCSC’s Genome browser and Stanford’s GREAT analysis were used to identify gene promoters. The commonality among affected genes was confirmed by Western blot. We observed nine significantly hypomethylated regions, two being significantly hypomethylated for both PE and IGUR. Western blot confirmed differential protein expression of commonly regulated genes. We conclude that despite the uniqueness of methylation profiles for PE and IUGR, the similarity of some methylation alterations in pathologies could explain the clinical similarities observed with these obstetric complications. These results also provide insight into the genetic similarity between PE and IUGR and suggest possible gene candidates plausibly involved in the onset of both conditions.

Receptors for advanced glycation end-products (RAGE) are multi-ligand cell surface receptors of the immunoglobin superfamily prominently expressed by lung epithelium. Previous experiments demonstrated that over-expression of RAGE by murine alveolar epithelium throughout embryonic development causes neonatal lethality coincident with significant lung hypoplasia. In the current study, we evaluated the expression of NKX2.1 (also referred to as TTF-1), a homeodomain-containing transcription factor critical for branching morphogenesis, in mice that differentially expressed RAGE. We also contextualized NKX2.1 expression with the abundance of FoxA2, a winged double helix DNA binding protein that influences respiratory epithelial cell differentiation and surfactant protein expression. Conditional RAGE over-expression was induced in mouse lung throughout gestation (embryonic day E0–18.5), as well as during the critical saccular period of development (E15.5–18.5), and analyses were conducted at E18.5. Histology revealed markedly less lung parenchyma beginning in the canalicular stage of lung development and continuing throughout the saccular period. We discovered consistently decreased expression of both NKX2.1 and FoxA2 in lungs from transgenic (TG) mice compared to littermate controls. We also observed diminished surfactant protein C in TG mice, suggesting possible hindered differentiation and/or proliferation of alveolar epithelial cells under the genetic control of these two critical transcription factors. These results demonstrate that RAGE must be specifically regulated during lung formation. Perturbation of epithelial cell differentiation culminating in respiratory distress and perinatal lethality may coincide with elevated RAGE expression in the lung parenchyma.

Intrauterine growth restriction (IUGR) is a disease affecting 10% of all pregnancies. IUGR is associated with maternal, fetal, and placental abnormalities, but studies investigating the inducibility of IUGR by secondhand smoke (SHS) are limited. The mTOR pathway regulates protein expression and cell growth. Disrupted mTOR signaling and apoptosis are associated with the development of several obstetric complications including IUGR. We tested the hypothesis that SHS exposure disrupts the mTOR pathway and apoptosis in mice. C57Bl6 mice were exposed to SHS for 4 days from day 14 to day 17 of gestation (dGA). At the time of necropsy (18 dGA) placental and fetal weights were recorded and tissues were immediately frozen for immunoblot analysis. Mice exposed to SHS demonstrated a significant reduction in fetal weight (7.35‐fold; p≤0.0001) and placental weight (1.13‐fold; p≤0.0001) compared to controls. Significant decreases were observed for placental activation of mTOR (2.1‐fold; p<0.03), p70 (1.9‐fold; p,0.03) and 4EBP1 (1.3‐fold (p<0.03) in IUGR placentas compared to controls. Increased placental active caspase 3 (1.1‐fold; p<0.04) and the antiapoptotic placental XIAP protein (2.2‐fold; p<0.03) were also detected during SHS treatment compared to controls. We conclude that decreased mTOR pathway is associated with lessened fetal and placental size during SHS induced IUGR. This decrease was associated with increased caspase 3 and that elevated XIAP protein may signify an attempted protective mechanism that counters increased apoptosis observed in the SHS‐induced IUGR placenta. These studies provide insight into tobacco‐mediated IUGR development and clarify avenues that may be helpful in the alleviation of placental complications.

Inflammation underpins pulmonary disease progression during tobacco smoke exposure that may culminate in irreversdible pulmonary remodeling. While primary smoke poses notable risk, nearly half of the US population is also at risk due to exposure to secondhand smoke (SHS). In the present study, we assessed a potential role for RAGE, a cell‐surface pattern recognition receptor implicated in pro‐inflammatory signaling, following exposure to SHS. Specifically, we used wild type, RAGE null, and lung‐specific RAGE overexpressing transgenic (TG) mice and evaluated the elaboration of inflammatory mediators in bronchoalveolar lavage fluid (BALF). Select mice were administered semi‐synthetic glycosaminoglycan ethers (SAGEs), a family of anionic, partially lipophilic sulfated polysaccharide derivatives known to inhibit RAGE signaling. Mice were exposed to room air (RA) or SHS from three Kentucky 3R4F research cigarettes via a nose‐only delivery system (Sireq Scientific, Montreal, Canada) five days a week and ip injections of PBS or SAGE (a 30mg/kg body weight) occurred three times per week from PN40 until sacrifice date on PN70. RAGE mRNA and protein expression was elevated in wild type and TG mice following SHS exposure and no expression was detected in RAGE nulls. BALF analyses revealed RAGE‐mediated control of leukocyte extravasation and a multiplex cytokine array confirmed a role for RAGE in the coordination of pro‐inflammatory chemokine/cytokine secretion. Among other mediators, TNF‐a, MIP‐2, and IL‐1b were each differentially secreted by lung tissue following SHS exposure and concentrations were significantly decreased in BALF from exposed RAGE null or wild type mice concomitantly administered SAGEs. In summary, inflammatory responses induced by SHS exposure were influenced by the availability of RAGE, as evidenced by RAGE nulls and SAGE treatment. These data reveal fascinating data suggesting the utility of RAGE abrogation in lessening smoke‐induced pulmonary exacerbations.

Receptors for advanced glycation end‐products (RAGE) are multi‐ligand cell surface receptors of the immunoglobin superfamily prominently expressed by lung epithelium. Previous experiments demonstrate that over‐expression of RAGE by murine alveolar epithelium throughout embryonic development causes neonatal lethality coincident with significant lung hypoplasia. In the current study, we evaluated the expression of TTF‐1, a homeodomain‐containing transcription factor critical for branching morphogeneis, in mice that differentially expressed RAGE. We also contexualized TTF‐1 expression with the abundance of FoxA2, a winged double helix DNA binding protein that influences respiratory epithelial cell differentiation, and surfactant proteins. Conditional RAGE over‐expression was induced in mice throughout gestation (embryonic day (E)0‐18.5) as well as during the critical saccular period (E15.5‐18.5) of development and analyses were conducted on E18.5 lung tissue. Histology revealed marked loss of lung tissue beginning in the canalicular stage of lung development and continuing throughout the saccular period. We discovered consistently decreased expression for both TTF‐1 and FoxA2 in lungs from TG mice compared to age‐matched controls. We also clarified diminished surfactant protein abundance in TG mice, suggesting possible hindered differentiation and/or proliferation of alveolar epithelial cells under the genetic control of these two critical transcription factors. These results demonstrate that RAGE must be specifically regulated during lung formation and that perturbation of epithelial cell differentiation culminating in respiratory distress and perinatal lethality may coincide with elevated RAGE expression in the lung parenchyma.