Emese Pinter

Arachidonic acid prevents hyperglycemia-associated yolk sac damage and embryopathy

Light microscopic, electron microscopic, and morphometric studies were performed on rat conceptuses cultured between day 10 and day 12 in normal, hyperglycemic, arachidonic acid-supplemented normal, and arachidonic acid-supplemented hyperglycemic rat serum. The results were compared with those of 12-day-old conceptuses grown in utero. No major differences were observed between in vivo and in vitro control conceptuses. Arachidonic acid supplementation of control culture medium resulted in an improvement of conceptus development. Addition of 20 micrograms/ml of arachidonic acid to an otherwise teratogenic hyperglycemic serum medium (950 mg/dl of D-glucose) prevented the malformations induced by hyperglycemic conditions: open neural tube, advanced neuropil formation in the neuroepithelium, significant reduction of rough endoplasmic reticulum, decreased size and number of lipid droplets, and increased number of lysosome-like structures in the visceral endodermal yolk sac cells.

Elevated glucose inhibits VEGF-A-mediated endocardial cushion formation: modulation by PECAM-1 and MMP-2

Atrioventricular (AV) septal defects resulting from aberrant endocardial cushion (EC) formation are observed at increased rates in infants of diabetic mothers. EC formation occurs via an epithelial-mesenchymal transformation (EMT), involving transformation of endocardial cells into mesenchymal cells, migration, and invasion into extracellular matrix. Here, we report that elevated glucose inhibits EMT by reducing myocardial vascular endothelial growth factor A (VEGF-A). This effect is reversed with exogenous recombinant mouse VEGF-A165, whereas addition of soluble VEGF receptor-1 blocks EMT. We show that disruption of EMT is associated with persistence of platelet endothelial cell adhesion molecule-1 (PECAM-1) and decreased matrix metalloproteinase-2 (MMP-2) expression. These findings correlate with retention of a nontransformed endocardial sheet and lack of invasion. The MMP inhibitor GM6001 blocks invasion, whereas explants from PECAM-1 deficient mice exhibit MMP-2 induction and normal EMT in high glucose. PECAM-1–negative endothelial cells are highly motile and express more MMP-2 than do PECAM-1–positive endothelial cells. During EMT, loss of PECAM-1 similarly promotes single cell motility and MMP-2 expression. Our findings suggest that high glucose-induced inhibition of AV cushion morphogenesis results from decreased myocardial VEGF-A expression and is, in part, mediated by persistent endocardial cell PECAM-1 expression and failure to up-regulate MMP-2 expression.

A null mutation of Hhex results in abnormal cardiac development,defective vasculogenesis and elevated Vegfa levels

The homeobox gene Hhex has recently been shown to be essential for normal liver, thyroid and forebrain development. Hhex–/– mice die by mid-gestation (E14.5) and the cause of their early demise remains unclear. Because Hhex is expressed in the developing blood islands at E7.0 in the endothelium of the developing vasculature and heart at E9.0-9.5, and in the ventral foregut endoderm at E8.5-9.0, it has been postulated to play a critical role in heart and vascular development. We show here, for the first time, that a null mutation of Hhex results in striking abnormalities of cardiac and vascular development which include: (1) defective vasculogenesis, (2) hypoplasia of the right ventricle, (3) overabundant endocardial cushions accompanied by ventricular septal defects, outflow tract abnormalities and atrio-ventricular (AV) valve dysplasia and (4) aberrant development of the compact myocardium. The dramatic enlargement of the endocardial cushions in the absence of Hhex is due to decreased apoptosis and dysregulated epithelial-mesenchymal transformation (EMT). Interestingly, vascular endothelial growth factor A (Vegfa) levels in the hearts of Hhex–/– mice were elevated as much as three-fold between E9.5 and E11.5, and treatment of cultured Hhex–/– AV explants with truncated soluble Vegfa receptor 1, sFlt-1, an inhibitor of Vegf signaling, completely abolished the excessive epithelial-mesenchymal transformation seen in the absence of Hhex. Therefore, Hhex expression in the ventral foregut endoderm and/or the endothelium is necessary for normal cardiovascular development in vivo, and one function of Hhex is to repress Vegfa levels during development.

Hyperglycemia-Induced Vasculopathy in the Murine Conceptus Is Mediated via Reductions of VEGF-A Expression and VEGF Receptor Activation

Major congenital malformations, including those affecting the cardiovascular system, remain the leading cause of mortality and morbidity in infants of diabetic mothers. Interestingly, targeted mutations of several genes (including VEGF and VEGF receptors) and many teratogenic agents (including excess D-glucose) that give rise to embryonic lethal phenotypes during organogenesis are associated with a failure in the formation and/or maintenance of a functional vitelline circulation. Given the similarities in the pathology of the abnormal vitelline circulation in many of these conditions, we hypothesized that the hyperglycemic insult present in diabetes could cause the resultant abnormalities in the vitelline circulation by affecting VEGF/VEGF receptor signaling pathway(s). In this study we report that hyperglycemic insult results in reduced levels of VEGF-A in the conceptus, which in turn, leads to abnormal VEGF receptor signaling, ultimately resulting in embryonic (vitelline) vasculopathy. These findings and our observation that addition of exogenous rVEGF-A(165) within a defined concentration range blunts the hyperglycemia-induced vasculopathy in the conceptus support the concept that VEGF levels can be modulated by glucose levels. In addition, these findings may ultimately lead to novel therapeutic approaches for the treatment of selected congenital cardiovascular abnormalities associated with diabetes.

Hyperglycemia-Induced Vasculopathy in the Murine Vitelline Vasculature: Correlation with PECAM-1/CD31 Tyrosine Phosphorylation State

Maternal diabetes mellitus is associated with an increased incidence of congenital abnormalities as well as embryonic and perinatal lethality. In particular, a wide range of cardiovascular abnormalities have been noted in children of diabetic mothers and in the offspring of diabetic animals. The vascular system is the first organ system to develop in the embryo and is critical for normal organogenesis. The organization of mesodermal cells into endothelial and hematopoietic cells and into a complex vascular system is, in part, mediated by a series of specific cell-cell, cell-extracellular matrix, and cell-factor interactions. PECAM-1 expression has been observed during the earliest stages of vasculogenesis, and changes in PECAM-1 tyrosine phosphorylation have been associated with endothelial cell migration, vasculogenesis, and angiogenesis both in vitro and in vivo. In this report we demonstrate that exposure to hyperglycemia during gastrulation causes yolk sac and embryonic vasculopathy in cultured murine conceptuses and in the conceptuses of streptozotocin-induced diabetic pregnant mice. In addition, we correlate the presence of yolk sac and embryonic vasculopathy with the failure of PECAM-1 tyrosine dephosphorylation during the formation of blood islands/vessels from clusters of extra-embryonic and embryonic angioblasts in the murine conceptus using both in vitro and in vivo models. The importance of these findings in the development of vasculopathy in the offspring of diabetic mothers and the potential effects and benefits of glucose regulation during the periods of vasculogenesis/angiogenesis in embryonic development are discussed.

Nitric oxide modulates murine yolk sac vasculogenesis and rescues glucose induced vasculopathy

Nitric oxide (NO) has been demonstrated to mediate events during ovulation, pregnancy, blastocyst invasion and preimplantation embryogenesis. However, less is known about the role of NO during postimplantation development. Therefore, in this study, we explored the effects of NO during vascular development of the murine yolk sac, which begins shortly after implantation. Establishment of the vitelline circulation is crucial for normal embryonic growth and development. Moreover, functional inactivation of the endodermal layer of the yolk sac by environmental insults or genetic manipulations during this period leads to embryonic defects/lethality, as this structure is vital for transport, metabolism and induction of vascular development. In this study, we describe the temporally/spatially regulated distribution of nitric oxide synthase (NOS) isoforms during the three stages of yolk sac vascular development (blood island formation, primary capillary plexus formation and vessel maturation/remodeling) and found NOS expression patterns were diametrically opposed. To pharmacologically manipulate vascular development, an established in vitro system of whole murine embryo culture was employed. During blood island formation, the endoderm produced NO and inhibition of NO (L-NMMA) at this stage resulted in developmental arrest at the primary plexus stage and vasculopathy. Furthermore, administration of a NO donor did not cause abnormal vascular development; however, exogenous NO correlated with increased eNOS and decreased iNOS protein levels. Additionally, a known environmental insult (high glucose) that produces reactive oxygen species (ROS) and induces vasculopathy also altered eNOS/iNOS distribution and induced NO production during yolk sac vascular development. However, administration of a NO donor rescued the high glucose induced vasculopathy, restored the eNOS/iNOS distribution and decreased ROS production. These data suggest that NO acts as an endoderm-derived factor that modulates normal yolk sac vascular development, and decreased NO bioavailability and NO-mediated sequela may underlie high glucose induced vasculopathy.

Review Article : The Yolk Sac Theory Closing the Circle on Why Diabetes-Associated Malformations Occur

The purpose of this article is to examine the role of yolk sac failure during organogenesis in the development of diabetes-associated embryopathy. The current literature regarding congenital malformations in diabetic pregnancies was reviewed to elucidate the precise role of the yolk sac in embryonic development and the relation between yolk sac injury and embryopathy. We and others have demonstrated that hyperglycemia produces a teratogenic effect during organogenesis. In addition, we have shown that the yolk sac appears to be the target site of injury induced by hyperglycemia. We have also presented evidence that cell membrane dysfunction leads to failed vitelline vessel formation and that arachidonic acid supplementation prevents many of the morphologic and biochemical alterations observed under hyperglycemic conditions. These data strongly support the teratogenic effect of hyperglycemia, the arachidonic acid deficiency state, the resultant maldevelopment of vitelline vessels, and the ability to prevent these changes by arachidonic acid supplementation. These studies have made significant inroads in explaining why diabetes-associated anomaties occur, and suggest a potential future role for prophylaxis against these organogenetic malformations using dietary polyunsaturated fatty acid supplementation. (J Soc Gynecol Invest 1994;1:3–13)OBJECTIVE: The purpose of this article is to examine the role of yolk sac failure during organogenesis in the development of diabetes-associated embryopathy. METHODS: The current literature regarding congenital malformations in diabetic pregnancies was reviewed to elucidate the precise role of the yolk sac in embryonic development and the relation between yolk sac injury and embryopathy. RESULTS: We and others have demonstrated that hyperglycemia produces a teratogenic effect during organogenesis. In addition, we have shown that the yolk sac appears to be the target site of injury induced by hyperglycemia. We have also presented evidence that cell membrane dysfunc tion leads to failed vitelline vessel formation and that arachidonic acid supplementation prevents many of the morphologic and biochemical alterations observed under hyperglycemic conditions. CONCLUSIONS: These data strongly support the teratogenic effect of hyperglycemia, the arachi donic acid deficiency state, the resultant maldevelopment of vitelline vessels, and the ability to prevent these changes by arachidonic acid supplementation. These studies have made significant inroads in explaining why diabetes-associated anomalies occur, and suggest a potential future role for prophylaxis against these organogenetic malformations using dietary polyunsaturated fatty acid supplementation. (J Soc Gynecol Invest 1994;1:3-13)

Fatty acid content of yolk sac and embryo in hyperglycemia-induced embryopathy and effect of arachidonic acid supplementation

Using the postimplantation rat conceptus model, we analyzed with gas-liquid chromatography, the fatty acid composition in major lipid groups (phospholipids, triglycerides, nonesterified fatty acids, and cholesterol esters) of yolk sacs and embryos cultured for 48 hours under control, hyperglycemic, and arachidonic acid-supplemented hyperglycemic conditions. In all experimental conditions the yolk sacs had greater fatty acid content than the embryos in all lipid groups except in nonesterified fatty acids. The fatty acid level in embryonic nonesterified fatty acids was significantly higher (p less than 0.05) in hyperglycemia-exposed embryos than found with arachidonic acid supplementation. Total yolk sac triglycerides were greater with added glucose (p less than 0.05) than with the addition of arachidonic acid to the same medium. Oleic acid, a fatty acid associated with essential fatty acid deficiency, was increased in the embryonic phospholipids and nonesterified fatty acids of conceptuses exposed to excess glucose, as well as in the culture media of this group, compared with the control or arachidonic acid-supplemented, hyperglycemic group (p less than 0.05). The results of this study demonstrate that diabetes-related embryopathy is associated with quantitative and qualitative abnormalities in major lipid groups. Furthermore, the elevation in embryonic oleic acid level suggests that the teratogenic mechanism could be related to a deficiency in essential fatty acids. The pattern of essential fatty acid deficiency and embryopathy was preventable with arachidonic acid supplementation in this experimental model.

A hypothesis concerning the general basis of organogenetic congenital anomalies

Evidence supports the idea that it is the degree of metabolic imbalance present in diabetic gravid women during the period of organogenesis that accounts for organogenetic congenital defects. In light of the proved and inferred metabolic instability occurring during early pregnancy, we propose that metabolic imbalances may occur that result in organogenetic congenital defects in offspring of apparently normal gravid women.

Maternal Diabetes: Effects on Embryonic Vascular Development—A Vascular Endothelial Growth Factor-A-mediated Process

Major congenital malformations, many of which result from abnormal cardiovascular patterning, remain the leading cause in infant mortality and morbidity. Targeted mutations of several genes (including VEGF and VEGF receptors) and certain teratogenic agents (including excess α-D-glucose) give rise to embryonic lethal phenotypes associated with failure in the formation of a functional vitelline circulation and aberrant organogenesis. Our work to date has demonstrated that yolk sac vasculopathy and failure of endocardial cushion epithelial-mesenchymal transformation occurs in hyperglycemic conditions in murine whole conceptus culture and in embryos from streptozotocin-induced diabetic mice. These cardiovascular abnormalities are associated with changes in expression and phosphorylation state of adhesion molecules such as platelet endothelial growth factor-1 and expression of growth factors such as vascular endothelial growth factor (VEGF-A). Further understanding of the effects of maternal diabetes on yolk sac and embryonic vasculogenesis/angiogenesis and organogenesis may lead to novel approaches in treating and preventing major birth defects.