Xiao-yu Wang

A New Oxidative Stress Model, 2,2-Azobis(2-Amidinopropane) Dihydrochloride Induces Cardiovascular Damages in Chicken Embryo

It is now well established that the developing embryo is very sensitive to oxidative stress, which is a contributing factor to pregnancy-related disorders. However, little is known about the effects of reactive oxygen species (ROS) on the embryonic cardiovascular system due to a lack of appropriate ROS control method in the placenta. In this study, a small molecule called 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH), a free radicals generator, was used to study the effects of oxidative stress on the cardiovascular system during chick embryo development. When nine-day-old (stage HH 35) chick embryos were treated with different concentrations of AAPH inside the air chamber, it was established that the LD50 value for AAPH was 10 µmol/egg. At this concentration, AAPH was found to significantly reduce the density of blood vessel plexus that was developed in the chorioallantoic membrane (CAM) of HH 35 chick embryos. Impacts of AAPH on younger embryos were also examined and discovered that it inhibited the development of vascular plexus on yolk sac in HH 18 embryos. AAPH also dramatically repressed the development of blood islands in HH 3+ embryos. These results implied that AAPH-induced oxidative stress could impair the whole developmental processes associated with vasculogenesis and angiogenesis. Furthermore, we observed heart enlargement in the HH 40 embryo following AAPH treatment, where the left ventricle and interventricular septum were found to be thickened in a dose-dependent manner due to myocardiac cell hypertrophy. In conclusion, oxidative stress, induced by AAPH, could lead to damage of the cardiovascular system in the developing chick embryo. The current study also provided a new developmental model, as an alternative for animal and cell models, for testing small molecules and drugs that have anti-oxidative activities.

Excess ROS induced by AAPH causes myocardial hypertrophy in the developing chick embryo

BACKGROUND The developing embryo is very sensitive to oxidative stress and excess reactive oxygen species (ROS) generation is often associated with cardiovascular malformation. However, little is known about the adverse effects of ROS during heart morphogenesis, especially during the formation of the atria and ventricles. METHODS AND RESULTS We have treated early chick embryos with 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH) to generate free radicals in the developing heart. We established that excess ROS induced by AAPH caused cardiomegaly to develop in 4-, 14- and 17-day-old embryos. The cardiomyocytes of these AAPH-treated hearts were hypertrophic, in both the compact and trabeculated myocardium. The weight of these hearts was also significantly increased in an AAPH dose-dependent fashion. We examined and compared the functions of the AAPH-treated and untreated hearts by echocardiography and determined that the ejection fraction was shortened. BrdU incorporation assay was performed and revealed that cell proliferation was not the main cause of cardiomegaly. However, we established that the cardiomyocytes exposed to excess ROS were distinctively larger than control cardiomyocytes - indicting that cardiomegaly was attributed to hypertrophy. We have also found that excess ROS inhibited Wnt signaling but enhanced VEGF signaling. Consequently, this promoted angiogenesis and caused larger coronary arteries to develop in the AAPH-treated hearts. CONCLUSIONS We have demonstrated that cardiomyocyte hypertrophy and changes in Wnt and VEGF signaling were the main contributing factors in the development of cardiomegaly induced by oxidative stress.

Investigating the Mechanism of Hyperglycemia-Induced Fetal Cardiac Hypertrophy

Hyperglycemia in diabetic mothers enhances the risk of fetal cardiac hypertrophy during gestation. However, the mechanism of high-glucose-induced cardiac hypertrophy is not largely understood. In this study, we first demonstrated that the incidence rate of cardiac hypertrophy dramatically increased in fetuses of diabetic mothers using color ultrasound examination. In addition, human fetal cardiac hypertrophy was successfully mimicked in a streptozotocin (STZ)-induced diabetes mouse model, in which mouse cardiac hypertrophy was diagnosed using type-M ultrasound and a histological assay. PH3 immunofluorescent staining of mouse fetal hearts and in vitro-cultured H9c2 cells indicated that cell proliferation decreased in E18.5, E15.5 and E13.5 mice, and cell apoptosis in H9c2 cells increased in the presence of high glucose in a dose-dependent manner. Next, we found that the individual cardiomyocyte size increased in pre-gestational diabetes mellitus mice and in response to high glucose exposure. Meanwhile, the expression of β-MHC and BMP-10 was up-regulated. Nkx2.5 immunofluorescent staining showed that the expression of Nkx2.5, a crucial cardiac transcription factor, was suppressed in the ventricular septum, left ventricular wall and right ventricular wall of E18.5, E15.5 and E13.5 mouse hearts. However, cardiac hypertrophy did not morphologically occur in E13.5 mouse hearts. In cultured H9c2 cells exposed to high glucose, Nkx2.5 expression decreased, as detected by both immunostaining and western blotting, and the expression of KCNE1 and Cx43 was also restricted. Taken together, alterations in cell size rather than cell proliferation or apoptosis are responsible for hyperglycemia-induced fetal cardiac hypertrophy. The aberrant expression of Nkx2.5 and its regulatory target genes in the presence of high glucose could be a principal component of pathogenesis in the development of fetal cardiac hypertrophy.

Autophagy functions on EMT in gastrulation of avian embryo.

Autophagy is important for cell renewing for its contribution to the degradation of bulk cytoplasm, long-lived proteins, and entire organelles and its role in embryonic development is largely unknown. In our study, we investigated the function of autophagy in gastrulation of the chick embryo using both in vivo and in vitro approaches, especially in the EMT process, and we found that autophagy gene Atg7 was expressed on the apical side of the ectoderm and endoderm. Over-expression of Atg7 could enhance the expression of Atg8 and the E-cadherin, the latter of which is a crucial marker of the EMT process. We also found that the disturbance of autophagy could retard the development of chick embryos in HH4 with shorter primitive steak than that in the control group, which is a newly formed structure during EMT process. So we assumed that autophagy could affect EMT process by adhesion molecule expression. Moreover, more molecules, such as slug, chordin, shh et., which were all involved in EMT process, were detected to address the mechanism of this phenomena. We established that the inhibition of autophagy could cause developmental delay by affecting EMT process in gastrulation of chick embryos.

Dimethyl phenyl piperazine iodide (DMPP) induces glioma regression by inhibiting angiogenesis

1,1-Dimethyl-4-phenyl piperazine iodide (DMPP) is a synthetic nicotinic acetylcholine receptor (nAChR) agonist that could reduce airway inflammation. In this study, we demonstrated that DMPP could dramatically inhibit glioma size maintained on the chick embryonic chorioallantoic membrane (CAM). We first performed MTT and BrdU incorporation experiments on U87 glioma cells in vitro to understand the mechanism involved. We established that DMPP did not significantly affect U87 cell proliferation and survival. We speculated that DMPP directly caused the tumor to regress by affecting the vasculature in and around the implanted tumor on our chick CAM model. Hence, we conducted detailed analysis of DMPPs inhibitory effects on angiogenesis. Three vasculogenesis and angiogenesis in vivo models were used in the study which included (1) early chick blood islands formation, (2) chick yolk-sac membrane (YSW) and (3) CAM models. The results revealed that DMPP directly suppressed all developmental stages involved in vasculogenesis and angiogenesis - possibly by acting through Ang-1 and HIF-2α signaling. In sum, our results show that DMPP could induce glioma regression grown on CAM by inhibiting vasculogenesis and angiogenesis.

High glucose environment inhibits cranial neural crest survival by activating excessive autophagy in the chick embryo

High glucose levels induced by maternal diabetes could lead to defects in neural crest development during embryogenesis, but the cellular mechanism is still not understood. In this study, we observed a defect in chick cranial skeleton, especially parietal bone development in the presence of high glucose levels, which is derived from cranial neural crest cells (CNCC). In early chick embryo, we found that inducing high glucose levels could inhibit the development of CNCC, however, cell proliferation was not significantly involved. Nevertheless, apoptotic CNCC increased in the presence of high levels of glucose. In addition, the expression of apoptosis and autophagy relevant genes were elevated by high glucose treatment. Next, the application of beads soaked in either an autophagy stimulator (Tunicamycin) or inhibitor (Hydroxychloroquine) functionally proved that autophagy was involved in regulating the production of CNCC in the presence of high glucose levels. Our observations suggest that the ERK pathway, rather than the mTOR pathway, most likely participates in mediating the autophagy induced by high glucose. Taken together, our observations indicated that exposure to high levels of glucose could inhibit the survival of CNCC by affecting cell apoptosis, which might result from the dysregulation of the autophagic process.

Imidacloprid Exposure Suppresses Neural Crest Cells Generation during Early Chick Embryo Development

Imidacloprid is a neonicotinoid pesticide that is widely used in the control pests found on crops and fleas on pets. However, it is still unclear whether imidacloprid exposure could affect early embryo development-despite some studies having been conducted on the gametes. In this study, we demonstrated that imidacloprid exposure could lead to abnormal craniofacial osteogenesis in the developing chick embryo. Cranial neural crest cells (NCCs) are the progenitor cells of the chick cranial skull. We found that the imidacloprid exposure retards the development of gastrulating chick embryos. HNK-1, PAX7, and Ap-2α immunohistological stainings indicated that cranial NCCs generation was inhibited after imidacloprid exposure. Double immunofluorescent staining (Ap-2α and PHIS3 or PAX7 and c-Caspase3) revealed that imidacloprid exposure inhibited both NCC proliferation and apoptosis. In addition, it inhibited NCCs production by repressing Msx1 and BMP4 expression in the developing neural tube and by altering expression of EMT-related adhesion molecules (Cad6B, E-Cadherin, and N-cadherin) in the developing neural crests. We also determined that imidacloprid exposure suppressed cranial NCCs migration and their ability to differentiate. In sum, we have provided experimental evidence that imidacloprid exposure during embryogenesis disrupts NCCs development, which in turn causes defective cranial bone development.

Autophagy is involved in high glucose-induced heart tube malformation

Both pre-gestational and gestational diabetes have an adverse impact on heart development, but little is known about the influence on the early stage of heart tube formation. Using early gastrulating chick embryos, we investigated the influence of high glucose on the process of heart tube formation, specifically during the primary heart field phase. We demonstrated that high-glucose exposure resulted in 3 types of heart tube malformation: 1) ventricular hypertrophy, 2) ventricular hypertrophy with dextrocardia and 3) ventricular hypertrophy and dextrocardia with the fusion anomaly of a bilateral primary heart tube. Next, we found that these malformation phenotypes of heart tubes might mainly originate from the migratory anomaly of gastrulating precardiac mesoderm cells rather than cell proliferation in the developmental process of bilateral primary heart field primordia. The treatment of rapamycin (RAPA), an autophagy inducer, led to a similar heart tube malformation phenotype as high glucose. Additionally, high-glucose exposure promoted the expression of the key autophagy protein LC3B in early chick tissue. Atg7 is strongly expressed in the fusion site of bilateral primary heart tubes. All of these data imply that autophagy could be involved in the process of high-glucose-induced malformation of the heart tube.

PTEN is involved in modulation of vasculogenesis in early chick embryos

Summary PTEN is a tumor suppressor gene that is mutated and/or deleted in many types of tumor. This gene also plays a very distinct role in the early stages of embryonic development such as cell migration, proliferation and migration. Nevertheless, little is known of the function of PTEN in vasculogenesis during chick embryonic development. In this study, we used in situ hybridization to first demonstrate the expression pattern of PTEN during gastrulation. PTEN was found mainly expressed in the blood islands of area opaca, the neural tube and mesodermal structures. Overexpression of PTEN obstructed the epithelial–mesenchymal transition (EMT) process in the primitive streak. EMT is the first prerequisite required for the emigration of hemangioblasts during vasculogenesis. When PTEN expression was silenced, we observed that it produced an adverse effect on mesodermal cell emigration to the extra-embryonic blood islands. In addition, we also demonstrated that even if the perturbed-PTEN cells did not affect the formation of blood islands, migrant mesodermal cells overexpressing wt PTEN-GFP had difficulties integrating into the blood islands. Instead, these cells were either localized on the periphery of the blood islands or induced to differentiate into endothelial cells if they managed to integrate into blood islands. Development of the intra-embryonic primary vascular plexus was also affected by overexpression of PTEN. We proposed that it was elevated PTEN lipid phosphatase activity that was responsible for the morphogenetic defects induced by PTEN overexpression. In this context, we did not find PTEN affecting VEGF signaling. In sum, our study has provided evidence that PTEN is involved in vasculogenesis during the early stages of chick embryo development.

Slit/Robo1 signaling regulates neural tube development by balancing neuroepithelial cell proliferation and differentiation

Formation of the neural tube is the morphological hallmark for development of the embryonic central nervous system (CNS). Therefore, neural tube development is a crucial step in the neurulation process. Slit/Robo signaling was initially identified as a chemo-repellent that regulated axon growth cone elongation, but its role in controlling neural tube development is currently unknown. To address this issue, we investigated Slit/Robo1 signaling in the development of chick neCollege of Life Sciences Biocentre, University of Dundee, Dundee DD1 5EH, UKural tube and transgenic mice over-expressing Slit2. We disrupted Slit/Robo1 signaling by injecting R5 monoclonal antibodies into HH10 neural tubes to block the Robo1 receptor. This inhibited the normal development of the ventral body curvature and caused the spinal cord to curl up into a S-shape. Next, Slit/Robo1 signaling on one half-side of the chick embryo neural tube was disturbed by electroporation in ovo. We found that the morphology of the neural tube was dramatically abnormal after we interfered with Slit/Robo1 signaling. Furthermore, we established that silencing Robo1 inhibited cell proliferation while over-expressing Robo1 enhanced cell proliferation. We also investigated the effects of altering Slit/Robo1 expression on Sonic Hedgehog (Shh) and Pax7 expression in the developing neural tube. We demonstrated that over-expressing Robo1 down-regulated Shh expression in the ventral neural tube and resulted in the production of fewer HNK-1(+) migrating neural crest cells (NCCs). In addition, Robo1 over-expression enhanced Pax7 expression in the dorsal neural tube and increased the number of Slug(+) pre-migratory NCCs. Conversely, silencing Robo1 expression resulted in an enhanced Shh expression and more HNK-1(+) migrating NCCs but reduced Pax7 expression and fewer Slug(+) pre-migratory NCCs were observed. In conclusion, we propose that Slit/Robo1 signaling is involved in regulating neural tube development by tightly coordinating cell proliferation and differentiation during neurulation.