Supplementation with the Methyl Donor Betaine Prevents Congenital Defects Induced by Prenatal Alcohol Exposure.

BACKGROUND: Despite decades of public education about dire consequences of prenatal alcohol exposure (PAE), drinking alcohol during pregnancy remains prevalent. As high as 40% of live-born infants exposed to alcohol during gestation and diagnosed with fetal alcohol syndrome have congenital heart defects that can be life-threatening. In animal models, the methyl donor betaine, found in foods such as wheat bran, quinoa, beets, and spinach, ameliorated neurobehavioral deficits associated with PAE, but effects on heart development are unknown. METHODS: Previously, we modeled a binge drinking episode during the first trimester in avian embryos. Here, we investigated whether betaine could prevent adverse effects of alcohol on heart development. Embryos exposed to ethanol (EtOH) with and without an optimal dose of betaine (5 µM) were analyzed at late developmental stages. Cardiac morphology parameters were rapidly analyzed and quantified using optical coherence tomography. DNA methylation at early stages was detected by immunofluorescent staining for 5-methylcytosine in sections of embryos treated with EtOH or cotreated with betaine. RESULTS: Compared to EtOH-exposed embryos, betaine-supplemented embryos had higher late-stage survival rates and fewer gross head and body defects than seen after alcohol exposure alone. Betaine also reduced the incidence of late-stage cardiac defects such as absent vessels, abnormal atrioventricular (AV) valves, and hypertrophic ventricles. Furthermore, betaine cotreatment brought measurements of great vessel diameters, interventricular septum thickness, and AV leaflet volumes in betaine-supplemented embryos close to control values. Early-stage 5-methycytosine staining revealed that DNA methylation levels were reduced by EtOH exposure and normalized by co-administration with betaine. CONCLUSIONS: This is the first study demonstrating efficacy of the methyl donor betaine in alleviating cardiac defects associated with PAE. These findings highlight the therapeutic potential of low-concentration betaine doses in mitigating PAE-induced birth defects and have implications for prenatal nutrition policies, especially for women who may not be responsive to folate supplementation.

Using optical coherence tomography to rapidly phenotype and quantify congenital heart defects associated with prenatal alcohol exposure.

BACKGROUND: The most commonly used method to analyze congenital heart defects involves serial sectioning and histology. However, this is often a time-consuming process where the quantification of cardiac defects can be difficult due to problems with accurate section registration. Here we demonstrate the advantages of using optical coherence tomography, a comparatively new and rising technology, to phenotype avian embryo hearts in a model of fetal alcohol syndrome where a binge-like quantity of alcohol/ethanol was introduced at gastrulation. RESULTS: The rapid, consistent imaging protocols allowed for the immediate identification of cardiac anomalies, including ventricular septal defects and misaligned/missing vessels. Interventricular septum thicknesses and vessel diameters for three of the five outflow arteries were also significantly reduced. Outflow and atrioventricular valves were segmented using image processing software and had significantly reduced volumes compared to controls. This is the first study to our knowledge that has 3D reconstructed the late-stage cardiac valves in precise detail to examine their morphology and dimensions. CONCLUSIONS: We believe, therefore, that optical coherence tomography, with its ability to rapidly image and quantify tiny embryonic structures in high resolution, will serve as an excellent and cost-effective preliminary screening tool for developmental biologists working with a variety of experimental/disease models.

Ethanol exposure alters early cardiac function in the looping heart: a mechanism for congenital heart defects?

Alcohol-induced congenital heart defects are frequently among the most life threatening and require surgical correction in newborns. The etiology of these defects, collectively known as fetal alcohol syndrome, has been the focus of much study, particularly involving cellular and molecular mechanisms. Few studies have addressed the influential role of altered cardiac function in early embryogenesis because of a lack of tools with the capability to assay tiny beating hearts. To overcome this gap in our understanding, we used optical coherence tomography (OCT), a nondestructive imaging modality capable of micrometer-scale resolution imaging, to rapidly and accurately map cardiovascular structure and hemodynamics in real time under physiological conditions. In this study, we exposed avian embryos to a single dose of alcohol/ethanol at gastrulation when the embryo is sensitive to the induction of birth defects. Late-stage hearts were analyzed using standard histological analysis with a focus on the atrio-ventricular valves. Early cardiac function was assayed using Doppler OCT, and structural analysis of the cardiac cushions was performed using OCT imaging. Our results indicated that ethanol-exposed embryos developed late-stage valvuloseptal defects. At early stages, they exhibited increased regurgitant flow and developed smaller atrio-ventricular cardiac cushions, compared with controls (uninjected and saline-injected embryos). The embryos also exhibited abnormal flexion/torsion of the body. Our evidence suggests that ethanol-induced alterations in early cardiac function have the potential to contribute to late-stage valve and septal defects, thus demonstrating that functional parameters may serve as early and sensitive gauges of cardiac normalcy and abnormalities.

Deletion of HIF-1α partially rescues the abnormal hyaloid vascular system in Cited2 conditional knockout mouse eyes.

PURPOSE: Cited2 (CBP/p300-interacting transactivators with glutamic acid (E) and aspartic acid (D)-rich tail 2) is a member of a new family of transcriptional modulators. Cited2 null embryos exhibit hyaloid hypercellularity consisting of aberrant vasculature in the eye. The purpose of the study is to address whether abnormal lenticular development is a primary defect of Cited2 deletion and whether deletion of hypoxia inducible factor (HIF)-1α or an HIF-1α target gene, vascular endothelial growth factor (VEGF), could rescue abnormal hyaloid vascular system (HVS) in Cited2 deficient adult eyes. METHODS: Le-Cre specific Cited2 knockout (Cited2(CKO)) mice with or without deletion of HIF-1α or VEGF were generated by standard Cre-Lox methods. Eyes collected from six-eight weeks old mice were characterized by Real Time PCR and immunohistological staining. RESULTS: Cited2(CKO) mice had smaller lenses, abnormal lens stalk formation, and failed regression of the HVS in the adult eye. The eye phenotype had features similar to persistent hyperplastic primary vitreous (PHPV), a human congenital eye disorder leading to abnormal lenticular development. Deletion of HIF-1α or VEGF in Cited2 knockout eyes partially rescued the abnormal HVS but had no effect on the smaller lens and abnormal lens stalk differentiation. Intravitreal injection of Topotecan (TPT), a compound that inhibits HIF-1α expression, partially eliminated HVS defects in Cited2(CKO) lenses. CONCLUSIONS: Abnormal HVS is a primary defect in Cited2 knockout mice, resulting in part from dysregulated functions of HIF-1 and VEGF. The Cited2(CKO) mouse line could be used as a novel disease model for PHPV and as an in vivo model for testing potential HIF-1 inhibitors.

Induction of quinone reductase by tamoxifen or DPN protects against mammary tumorigenesis.

We have previously shown that estrogen receptor ß (ERß)-mediated up-regulation of quinone reductase (QR) is involved in the protection against estrogen-induced mammary tumorigenesis. Our present study provides evidence that the ERß agonist, 2,3-bis-(4-hydroxy-phenyl)-propionitrile (DPN), and the selective estrogen receptor modulator tamoxifen (Tam), inhibit estrogen-induced DNA damage and mammary tumorigenesis in the aromatase transgenic (Arom) mouse model. We also show that either DPN or Tam treatment increases QR levels and results in a decrease in ductal hyperplasia, proliferation, oxidative DNA damage (ODD), and an increase in apoptosis. To corroborate the role of QR, we provide additional evidence in triple transgenic MMTV/QR/Arom mice, wherein the QR expression is induced in the mammary glands via doxycycline, causing a decrease in ductal hyperplasia and ODD. Overall, we provide evidence that up-regulation of QR through induction by Tam or DPN can inhibit estrogen-induced ODD and mammary cell tumorigenesis, representing a novel mechanism of prevention against breast cancer. Thus, our data have important clinical implications in the management of breast cancer; our findings bring forth potentially new therapeutic strategies involving ERß agonists.

Optical coherence tomography captures rapid hemodynamic responses to acute hypoxia in the cardiovascular system of early embryos.

BACKGROUND: The trajectory to heart defects may start in tubular and looping heart stages when detailed analysis of form and function is difficult by currently available methods. We used a novel method, Doppler optical coherence tomography (OCT), to follow changes in cardiovascular function in quail embryos during acute hypoxic stress. Chronic fetal hypoxia is a known risk factor for congenital heart diseases (CHDs). Decreased fetal heart rates during maternal obstructive sleep apnea suggest that studying fetal heart responses under acute hypoxia is warranted. RESULTS: We captured responses to hypoxia at the critical looping heart stages. Doppler OCT revealed detailed vitelline arterial pulsed Doppler waveforms. Embryos tolerated 1 hr of hypoxia (5%, 10%, or 15% O(2) ), but exhibited changes including decreased systolic and increased diastolic duration in 5 min. After 5 min, slower heart rates, arrhythmic events and an increase in retrograde blood flow were observed. These changes suggested slower filling of the heart, which was confirmed by four-dimensional Doppler imaging of the heart itself. CONCLUSIONS: Doppler OCT is well suited for rapid noninvasive screening for functional changes in avian embryos under near physiological conditions. Analysis of the accessible vitelline artery sensitively reflected changes in heart function and can be used for rapid screening. Acute hypoxia caused rapid hemodynamic changes in looping hearts and may be a concern for increased CHD risk.

Klf15 deficiency is a molecular link between heart failure and aortic aneurysm formation.

Current therapies for diseases of heart muscle (cardiomyopathy) and aorta (aortopathy) include inhibitors of the renin-angiotensin system, beta-adrenergic antagonists, and the statin class of cholesterol-lowering agents. These therapies have limited efficacy, as adverse cardiovascular events continue to occur with some frequency in patients taking these drugs. Although cardiomyopathy and aortopathy can coexist in a number of conditions (for example, Marfan's syndrome, acromegaly, pregnancy, and aging), pathogenetic molecular links between the two diseases remain poorly understood. We reasoned that identification of common molecular perturbations in these two tissues could point to therapies for both conditions. Here, we show that deficiency of the transcriptional regulator Kruppel-like factor 15 (Klf15) in mice leads to both heart failure and aortic aneurysm formation through a shared molecular mechanism. Klf15 concentrations are markedly reduced in failing human hearts and in human aortic aneurysm tissues. Mice deficient in Klf15 develop heart failure and aortic aneurysms in a p53-dependent and p300 acetyltransferase-dependent fashion. KLF15 activation inhibits p300-mediated acetylation of p53. Conversely, Klf15 deficiency leads to hyperacetylation of p53 in the heart and aorta, a finding that is recapitulated in human tissues. Finally, Klf15-deficient mice are rescued by p53 deletion or p300 inhibition. These findings highlight a molecular perturbation common to the pathobiology of heart failure and aortic aneurysm formation and suggest that manipulation of KLF15 function may be a productive approach to treat these morbid diseases.

Expression of lymphatic markers during avian and mouse cardiogenesis.

The adult heart has been reported to have an extensive lymphatic system, yet the development of this important system during cardiogenesis is still largely unexplored. The nuclear-localized transcription factor Prox-1 identified a sheet of Prox-1-positive cells on the developing aorta and pulmonary trunk in avian and murine embryos just before septation of the four heart chambers. The cells coalesced into a branching lymphatic network that spread within the epicardium to cover the heart. These vessels eventually expressed the lymphatic markers LYVE-1, VEGFR-3, and podoplanin. Before the Prox-1-positive cells were detected in the mouse epicardium, LYVE-1, a homologue of the CD44 glycoprotein, was primarily expressed in individual epicardial cells. Similar staining patterns were observed for CD44 in avian embryos. The proximity of these LYVE-1/CD44-positive mesenchymal cells to Prox-1-positive vessels suggests that they may become incorporated into the lymphatics. Unexpectedly, we detected LYVE-1/PECAM/VEGFR-3-positive vessels within the embryonic and adult myocardium, which remained Prox-1/podoplanin-negative. Lymphatic markers were surprisingly found in adult rat and embryonic mouse epicardial cell lines, with Prox-1 also exhibiting nuclear-localized expression in primary cultures of embryonic avian epicardial cells. Our data identified three types of cells in the embryonic heart expressing lymphatic markers: (1) Prox-1-positive cells from an extracardiac source that migrate within the serosa of the outflow tract into the epicardium of the developing heart, (2) individual LYVE-1-positive cells in the epicardium that may be incorporated into the Prox-1-positive lymphatic vasculature, and (3) LYVE-1-positive cells/vessels in the myocardium that do not become Prox-1-positive even in the adult heart.

Expression of active Notch1 in avian coronary development.

Notch1 is an important regulator of intercellular interactions in cardiovascular development. We show that the nuclear-localized, cleaved and active form of Notch1, the Notch1 intracellular domain (N1ICD), appeared in mesothelial cells of the pro-epicardium during epicardial formation at looped heart stages. N1ICD was also present in mesothelial cells and mesenchymal cells specifically within the epicardium at sulcus regions. N1ICD-positive endothelial cells were detected within the nascent vessel plexus at the atrioventricular junction and within the compact myocardium (Hamburger and Hamilton stage [HH] 25-HH30). The endothelial cells expressing N1ICD were surrounded by N1ICD-positive smooth muscle cells after coronary orifice formation (HH32-HH35), while N1ICD expression was absent in the mesenchymal and mesothelial cells surrounding mature coronary vessels. We propose that differential activation of the hypoxia/HIF1-VEGF-Notch pathway may play a role in epicardial cell interactions that promote epicardial epithelial/mesenchymal transition and coronary progenitor cell differentiation during epicardial development and coronary vasculogenesis in particularly hypoxic sulcus regions.

Mouse and human phenotypes indicate a critical conserved role for ERK2 signaling in neural crest development.

Disrupted ERK1/2 (MAPK3/MAPK1) MAPK signaling has been associated with several developmental syndromes in humans; however, mutations in ERK1 or ERK2 have not been described. We demonstrate haplo-insufficient ERK2 expression in patients with a novel approximately 1 Mb micro-deletion in distal 22q11.2, a region that includes ERK2. These patients exhibit conotruncal and craniofacial anomalies that arise from perturbation of neural crest development and exhibit defects comparable to the DiGeorge syndrome spectrum. Remarkably, these defects are replicated in mice by conditional inactivation of ERK2 in the developing neural crest. Inactivation of upstream elements of the ERK cascade (B-Raf and C-Raf, MEK1 and MEK2) or a downstream effector, the transcription factor serum response factor resulted in analogous developmental defects. Our findings demonstrate that mammalian neural crest development is critically dependent on a RAF/MEK/ERK/serum response factor signaling pathway and suggest that the craniofacial and cardiac outflow tract defects observed in patients with a distal 22q11.2 micro-deletion are explained by deficiencies in neural crest autonomous ERK2 signaling.

HEXIM1 regulates 17beta-estradiol/estrogen receptor-alpha-mediated expression of cyclin D1 in mammary cells via modulation of P-TEFb.

Estrogen receptor alpha (ERalpha) plays a key role in mammary gland development and is implicated in breast cancer through the transcriptional regulation of genes linked to proliferation and apoptosis. We previously reported that hexamethylene bisacetamide inducible protein 1 (HEXIM1) inhibits the activity of ligand-bound ERalpha and bridges a functional interaction between ERalpha and positive transcription elongation factor b (P-TEFb). To examine the consequences of a functional HEXIM1-ERalpha-P-TEFb interaction in vivo, we generated MMTV/HEXIM1 mice that exhibit mammary epithelial-specific and doxycycline-inducible expression of HEXIM1. Increased HEXIM1 expression in the mammary gland decreased estrogen-driven ductal morphogenesis and inhibited the expression of cyclin D1 and serine 2 phosphorylated RNA polymerase II (S2P RNAP II). In addition, increased HEXIM1 expression in MCF-7 cells led to a decrease in estrogen-induced cyclin D1 expression, whereas down-regulation of HEXIM1 expression led to an enhancement of estrogen-induced cyclin D1 expression. Studies on the mechanism of HEXIM1 regulation on estrogen action indicated a decrease in estrogen-stimulated recruitment of ERalpha, P-TEFb, and S2P RNAP II to promoter and coding regions of ERalpha-responsive genes pS2 and CCND1 with increased HEXIM1 expression in MCF-7 cells. Notably, increased HEXIM1 expression decreased only estrogen-induced P-TEFb activity. Whereas there have been previous reports on HEXIM1 inhibition of P-TEFb activity, our studies add a new dimension by showing that E(2)/ER is an important regulator of the HEXIM1/P-TEFb functional unit in breast cells. Together, these studies provide novel insight into the role of HEXIM1 and ERalpha in mammary epithelial cell function.

Cited2 is required for the proper formation of the hyaloid vasculature and for lens morphogenesis.

Cited2 is a transcriptional modulator with pivotal roles in different biological processes. Cited2-deficient mouse embryos manifested two major defects in the developing eye. An abnormal corneal-lenticular stalk was characteristic of Cited2(-/-) developing eyes, a feature reminiscent of Peters' anomaly, which can be rescued by increased Pax6 gene dosage in Cited2(-/-) embryonic eyes. In addition, the hyaloid vascular system showed hyaloid hypercellularity consisting of aberrant vasculature, which might be correlated with increased VEGF expression in the lens. Deletion of Hif1a (which encodes HIF-1alpha) in Cited2(-/-) lens specifically eliminated the excessive accumulation of cellular mass and aberrant vasculature in the developing vitreous without affecting the corneal-lenticular stalk phenotype. These in vivo data demonstrate for the first time dual functions for Cited2: one upstream of, or together with, Pax6 in lens morphogenesis; and another in the normal formation of the hyaloid vasculature through its negative modulation of HIF-1 signaling. Taken together, our study provides novel mechanistic revelation for lens morphogenesis and hyaloid vasculature formation and hence might offer new insights into the etiology of Peters' anomaly and ocular hypervascularity.

Cited2 is required for fetal lung maturation.

Lung maturation at the terminal sac stage of lung development is characterized by a coordinated increase in terminal sac formation and vascular development in conjunction with the differentiation of alveolar type I and type II epithelial cells. The Cited2-Tcfap2a/c complex has been shown to activate transcription of Erbb3 and Pitx2c during mouse development. In this study, we show that E17.5 to E18.5 Cited2-null lungs had significantly reduced terminal sac space due to an altered differentiation of type I and type II alveolar epithelial cells. In addition, E17 Cited2-null lungs exhibited a decrease in the number of apoptotic cells, contributing to the loss in airspace. Consistent with the phenotype, genes associated with alveolar cell differentiation and survival were differentially expressed in Cited2-null fetal lungs compared to those of wild-type littermates. Moreover, expression of Cebpa, a key regulator of airway epithelial maturation, was significantly decreased in Cited2-null fetal lungs. Cited2 and Tcfap2c were present on the Cebpa promoter in E18.5 lungs to activate Cebpa transcription. We propose that the Cited2-Tcfap2c complex controls lung maturation by regulating Cebpa expression. Understanding the function of this complex may provide novel therapeutic strategies for patients with respiratory distress syndromes.

Cited2, a coactivator of HNF4alpha, is essential for liver development.

The transcriptional modulator Cited2 is induced by various biological stimuli including hypoxia, cytokines, growth factors, lipopolysaccharide (LPS) and flow shear. In this study, we report that Cited2 is required for mouse fetal liver development. Cited2(-/-) fetal liver displays hypoplasia with higher incidence of cell apoptosis, and exhibits disrupted cell-cell contact, disorganized sinusoidal architecture, as well as impaired lipid metabolism and hepatic gluconeogenesis. Furthermore, we demonstrated the physical and functional interaction of Cited2 with liver-enriched transcription factor HNF4alpha. Chromatin immunoprecipitation (ChIP) assays further confirmed the recruitment of Cited2 onto the HNF4alpha-responsive promoters and the reduced HNF4alpha binding to its target gene promoters in the absence of Cited2. Taken together, this study suggests that fetal liver defects in mice lacking Cited2 result, at least in part, from its defective coactivation function for HNF4alpha.

Apoptosis in the developing mouse heart.

Apoptosis occurs at high frequency in the myocardium of the developing avian cardiac outflow tract (OFT). Up- or down-regulating apoptosis results in defects resembling human conotruncal heart anomalies. This finding suggested that regulated levels of apoptosis are critical for normal morphogenesis of the four-chambered heart. Recent evidence supports an important role for hypoxia of the OFT myocardium in regulating cell death and vasculogenesis. The purpose of this study was to determine whether apoptosis in the outflow tract myocardium occurs in the mouse heart during developmental stages comparable to the avian heart and to determine whether differential hypoxia is also present at this site in the murine heart. Apoptosis was detected using a fluorescent vital dye, Lysotracker Red (LTR), in the OFT myocardium of the mouse starting at embryonic day (E) 12.5, peaking at E13.5-14.5, and declining thereafter to low or background levels by E18.5. In addition, high levels of apoptosis were detected in other cardiac regions, including the apices of the ventricles and along the interventricular sulcus. Apoptosis in the myocardium was detected by double-labeling with LTR and cardiomyocyte markers. Terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) and immunostaining for cleaved Caspase-3 were used to confirm the LTR results. At the peak of OFT apoptosis in the mouse, the OFT myocardium was relatively hypoxic, as indicated by specific and intense EF5 staining and HIF1alpha nuclear localization, and was surrounded by the developing vasculature as in the chicken embryo. These findings suggest that cardiomyocyte apoptosis is an evolutionarily conserved mechanism for normal morphogenesis of the outflow tract myocardium in avian and mammalian species.

Differential levels of tissue hypoxia in the developing chicken heart.

Tissue hypoxia plays a critical role in normal development, including cardiogenesis. Previously, we showed that oxygen concentration, as assessed by the hypoxia indicator EF5, is lowest in the outflow tract (OFT) myocardium of the developing chicken heart and may be regulating events in OFT morphogenesis. In this study, we identified additional areas of the embryonic chicken heart that were intensely positive for EF5 within the myocardium in discrete regions of the atrial wall and the interventricular septum (IVS). The region of the IVS that is EF5-positive includes a portion of the developing central conduction system identified by HNK-1 co-immunostaining. The EF5 positive tissues were also specifically positive for nuclear-localized hypoxia inducible factor 1alpha (HIF-1alpha), the oxygen-sensitive component of the hypoxia inducible factor 1 (HIF-1) heterodimer. The pattern of the most intensely EF5-stained myocardial regions of the atria and IVS resemble the pattern of the major coronary vessels that form in later stages within or immediately adjacent to these particular regions. These vessels include the sinoatrial nodal artery that is a branch of the right coronary artery within the atrial wall and the anterior/posterior interventricular vessels of the IVS. These findings indicate that a portion of the developing central conduction system and the patterning of coronary vessels may be subject to a level of regulation that is dependent on differential oxygen concentration within cardiac tissues and subsequent HIF-1 regulation of gene expression.

Dynamic patterns of apoptosis in the developing chicken heart.

The outflow tract (OFT) is abnormal in many congenital heart defects. One critical mechanism for morphogenesis of this complex structure is apoptosis. Chicken embryos (stages 19-38; ED4-10) stained with a fluorescent supravital lysosomal dye (LysoTracker Red; LTR) revealed the three-dimensional relationship between structural changes and apoptosis. The LTR staining peaked in the OFT myocardium at stages 27-32, consistent with our previous analyses using other apoptosis assays. While LTR stained under both the pulmonary artery and the aorta, it was most prevalent in the subaortic myocardium before its elimination. Furthermore, LTR staining was most abundant in the myocardium under intensely cytokeratin-positive, thick epicardium. These data support the hypothesis that temporally and spatially restricted apoptosis in the OFT myocardium allows the aorta and pulmonary artery to dock at the appropriate angle and level with the proper ventricle. These data also support a relationship between the differentiating epicardium and cardiomyocyte apoptosis.