The development of yolk sac vasculature

Although the human yolk sac resides outside of the embryo, similar to the rodent yolk sac, it plays an important role in early embryonic vasculogenesis. The murine yolk sac is derived from the same progenitor cells that produce the embryo. In mice, conceptus vasculogenesis starts with the emergence of vascular endothelial growth factor receptor-2-positive (Flk ⁺ ) cells in the yolk sac. These Flk1 ⁺ progenitor endothelial cells form blood islands that fuse to generate a primary capillary plexus at embryonic day 7.5. In addition, extraembryonic mesodermal cells proliferate to form angioblastic cords on embryonic day 7.5. At embryonic day 8.0, blood islands fuse and establish the primary capillary network, which intimately is associated with mural cells. By embryonic day 9.5, the capillary plexus has remodeled into a complex hierarchy of mature small and large vessels, and functional vitelline circulation is established. A critical number of Flk1 ⁺ cells and blood islands are crucial for normal vasculogenesis.

Vasculogenesis begins in the yolk sac before embryonic vasculogenesis and development of the cardiovascular system. In addition, the yolk sac and embryonic vasculatures are regulated by the same group of angiogenic and survival factors via common mechanisms. Therefore, the elucidation of the mechanism underlying hyperglycemia-induced yolk sac vasculopathy is important in the cause of diabetic embryopathy.

Maternal diabetes mellitus induces yolk sac structure failure and dysfunction

Experimental evidence has elucidated the precise role of the yolk sac in mammalian embryonic development and the relationship between yolk sac injury and embryopathy. The structures and prostaglandin E2 levels of human yolk sacs are altered by maternal diabetes mellitus. Studies have shown that yolk sac development morphologically is impaired under hyperglycemic conditions. For example, conceptuses exposed to excess glucose demonstrate decreased size and gross malformations. Furthermore, exposure to excess glucose causes the visceral yolk sac capillaries and vitelline vessels to become sparse, patchy, and not uniformly located. Under high glucose conditions, the visceral yolk sac endodermal cells have reduced numbers of rough endoplasmic reticulum, ribosomes, and mitochondria. These defects in yolk sac structures suggest that hyperglycemia during organogenesis has a primarily deleterious effect on yolk sac functions.

Hyperglycemic conditions also appear to affect the transport function of the yolk sac. For example, experiments that used horseradish peroxidase as a tracer protein to examine the transport function of the visceral endodermal yolk sac cells have shown that the cellular uptake of peroxidase is diminished in conceptuses that were cultured under hyperglycemic conditions. These findings indicate that hyperglycemia inhibits transport of nutrients from the yolk sac to the embryo. Coupled together with the experiments that demonstrate a deleterious effect of hyperglycemia on cell morphologic condition, these data suggest that yolk sac failure is associated with diabetic embryopathy.

Maternal diabetes mellitus induces yolk sac structure failure and dysfunction

Experimental evidence has elucidated the precise role of the yolk sac in mammalian embryonic development and the relationship between yolk sac injury and embryopathy. The structures and prostaglandin E2 levels of human yolk sacs are altered by maternal diabetes mellitus. Studies have shown that yolk sac development morphologically is impaired under hyperglycemic conditions. For example, conceptuses exposed to excess glucose demonstrate decreased size and gross malformations. Furthermore, exposure to excess glucose causes the visceral yolk sac capillaries and vitelline vessels to become sparse, patchy, and not uniformly located. Under high glucose conditions, the visceral yolk sac endodermal cells have reduced numbers of rough endoplasmic reticulum, ribosomes, and mitochondria. These defects in yolk sac structures suggest that hyperglycemia during organogenesis has a primarily deleterious effect on yolk sac functions.

Hyperglycemic conditions also appear to affect the transport function of the yolk sac. For example, experiments that used horseradish peroxidase as a tracer protein to examine the transport function of the visceral endodermal yolk sac cells have shown that the cellular uptake of peroxidase is diminished in conceptuses that were cultured under hyperglycemic conditions. These findings indicate that hyperglycemia inhibits transport of nutrients from the yolk sac to the embryo. Coupled together with the experiments that demonstrate a deleterious effect of hyperglycemia on cell morphologic condition, these data suggest that yolk sac failure is associated with diabetic embryopathy.

Maternal diabetes mellitus induces yolk sac vasculopathy

In mice, abnormal development and arrested development of the yolk sac vasculature on embryonic day 7.5 can result in congenital malformations in a wide variety of organs and tissues and embryonic lethality. The adverse effects of hyperglycemia on the yolk sac have been documented in maternal diabetic animal models and in vitro cultured rodent embryos. Under hyperglycemic conditions, development of the blood vessels in the yolk sac is disrupted, and the cellular structures in the vessels are altered. Conceptuses display various, profoundly abnormal yolk sac vasculature, with some completely devoid of vasculogenesis; others have a branched plexus with no apparent arborization or distinction of arteries and veins.

The adverse effects of hyperglycemia on yolk sac vasculature development can be characterized by arbitrarily assigning morphologic scores to individual vasculatures. Using this rating system, 1 group showed that the yolk sac vasculature score of the hyperglycemia group was significantly lower than that of the euglycemic group. Yolk sac vasculature morphologic scores were correlated inversely with embryonic malformation rates, such that the higher the score, the lower the rate of malformations, and vice versa.

Although the developing yolk sac contains a diverse cell population, evidence shows that vascular endothelial cells are the primary targets of hyperglycemic insults. Platelet-derived endothelial cell adhesion molecule, which is an endothelial cell marker, modulates endothelial cell migration, cell-cell adhesion, and in vitro and in vivo angiogenesis. Under hyperglycemic conditions, the presence of yolk sac vasculopathy is associated with the failure of Platelet-derived endothelial cell adhesion molecule tyrosine phosphorylation. Thus, hyperglycemia may impact vascular endothelial cell functions adversely, including apoptosis, proliferation, and differentiation through regulation of endothelial cell-specific cellular intermediates and signaling.

Molecular intermediates and signaling pathways contribute to maternal diabetes mellitus–induced yolk sac vasculopathy

Studies show that maternal diabetes mellitus induces yolk sac vasculopathy through 2 distinct sets of molecular events. In 1 set of events, hypoxia-inducible factor 1(HIF-1) and vascular endothelial growth factor (VEGF), which are 2 proteins that are typically active in normal vasculogenesis, are down-regulated by maternal diabetes mellitus. In another set of events, maternal diabetes mellitus induces activation of a key apoptosis-related kinase, known as apoptosis signal-regulating kinase 1 (ASK1), which increases induced nitric oxide synthase (iNOS) expression and the promotion of apoptosis. Inhibition of events downstream of ASK1 activation, such as c-Jun-N-terminal kinases (JNK1/2) signaling, abolishes maternal diabetes mellitus–induced vasculopathy. The protective effect of thioredoxin-1, an inhibitor of ASK1, on hyperglycemia-induced vasculopathy has been demonstrated. The elucidation of the mechanisms underlying hyperglycemia-induced yolk sac vasculopathy can aid in the development of preventative methods for maternal diabetes mellitus–induced cardiovascular defects in humans.

The role of HIF-1 in yolk sac vasculopathy

HIF-1 is a key transcriptional regulator for hypoxia regulation of embryonic vascular development. It is an oxygen-sensitive heterodimer that consists of a constitutively expressed HIF-1β subunit and an oxygen-regulated HIF-1α subunit. Regulation of HIF-1 activity depends on the degradation of the HIF-1α subunit in normoxic conditions. The molecular basis of HIF-1α degradation is the oxygen-dependent hydroxylation of at least 1 of the 2 proline residues in its oxygen-dependent degradation domain by specific prolylhydroxylases (PHD1, PHD2 and PHD3). In this form, HIF-1α binds to the von Hippel-Lindau tumor suppressor protein, which acts as an E3 ubiquitin ligase and targets HIF-1α for proteasomal degradation. During conditions of normoxia, HIF-1β is found in the nucleus; HIF-1α is cytoplasmic and rapidly degraded. Reduced oxygen levels during embryonic development permit the accumulation of HIF-1α protein in the cytoplasm. Subsequently, HIF-1α translocates to the nucleus, engages HIF-1β, and forms the HIF-1 complex that initiates transcription.

HIF-1 functions as a master regulator of angiogenesis by controlling the expression of multiple angiogenic growth factors. Maternal diabetes mellitus has been shown to reduce HIF-1α levels in the embryo, leading to vasculopathy. Maternal diabetes mellitus reduces the embryonic hypoxic environment-induced HIF-1α. AdCA5, an adenovirus encoding a constitutively active form of HIF-1α, blocks diabetes mellitus–induced vasculopathy, which demonstrates that HIF-1α reduction contributes to diabetes mellitus–induced vasculopathy. Mice that lack HIF-1 activity because of HIF-1α- or HIF-1β-null mutations develop extensive vascular defects, similar to those that have been observed in diabetic yolk sac vasculopathy, including inadequate vessel formation and aberrant vascular remodeling. HIF-1 deficiency also decreases cell survival, leading to abnormal vasculogenesis. In our previous study, we demonstrated that a decrease in HIF-1α expression is responsible for the VEGF reduction that is induced by maternal diabetes mellitus. This suggests that the HIF-1α VEGF signaling pathway plays a role in maternal diabetes mellitus–induced vasculopathy ( Figure 1 ).

Maternal diabetes mellitus induces yolk sac vasculopathy via reduction of hypoxia-inducible factor 1α

Under normoxic conditions, specific prolylhydroxylases induce oxygen-dependent hydroxylation of HIF-1α. HIF-1α then binds to the von Hippel-Lindau tumor suppressor protein, which acts as an E3 ubiquitin ligase and targets HIF-1α for proteasomal degradation. Under hypoxic conditions, HIF-1β translocates to the nucleus, engages HIF-1β, and forms the HIF-1 complex that initiates transcription of downstream genes, which include vascular endothelial growth factors. Maternal diabetes mellitus reduces HIF-1α levels by enhancing its degradation. The lack of HIF-1α leads to the development of extensive vascular defects, which is similar to diabetic yolk sac vasculopathy.

HIF-1 , hypoxia-inducible factor 1; PHDs , prolylhydroxylases; pVHL , von Hippel-Lindau tumor suppressor protein; VEGFs , vascular endothelial growth factors.

Dong. The role of yolk sac in diabetic embryopathy. Am J Obstet Gynecol 2016 .

The proapoptotic ASK1-JNK1/2 pathway

Apoptosis has been hypothesized as a primary mechanism of diabetes mellitus–induced birth defects. Under euglycemic conditions, very low basal levels of apoptosis are observed in the embryonic tissues during organogenesis (embryonic days 7-11). In contrast, compelling evidence demonstrates that maternal hyperglycemia enhances apoptosis in the embryonic days 7-11 embryonic tissues. However, the apoptotic mechanism in this disease process is not well understood. Evidence from clinical and experimental studies has revealed that maternal diabetes mellitus leads to an imbalance in intracellular reduction-oxidation (redox) homeostasis, resulting in intracellular oxidative stress. Recent studies have demonstrated that oxidative stress and endoplasmic reticulum (ER) stress are the main biochemical and molecular mechanisms underlying maternal diabetes mellitus– induced apoptosis.

JNK1/2 are proapoptotic factors that belong to the mitogen-activated protein kinase (MAPK) family. MAPKs are members of a complex superfamily of serine/threonine kinases that are activated in response to a variety of extracellular stimuli. The basic assembly of the MAPK signaling pathway is a 3-component module that involves sequential activation of MAPK kinase kinase (MAP3K), MAPK kinase (MAPKK), and MAPK. MAP3K phosphorylates and thereby activates MAPKK; activated MAPKK in turn phosphorylates and activates MAPK. Because the activation status of MAPKs depends largely on MAP3Ks, it is important to understand how MAP3Ks are regulated. Fourteen different MAP3Ks have been identified. Among them, several MAP3Ks (including ASK1, TAK1, and MLK3) are known to activate the JNK pathway in response to diverse stimuli. In our previous work, we indicated that, at a concentration of 800 nmol/L, an inhibitor of JNK1/2 (SP600125) significantly abrogated hyperglycemia-induced yolk sac vasculopathy in both morphologic score and vasculature morphologic condition, which strongly suggested that JNK1/2 activation plays an important role in hyperglycemia-induced yolk sac vasculopathy ( Figure 2 ).

Maternal diabetes mellitus induces endothelial progenitor apoptosis via apoptosis signal-regulating kinase 1 activation

Maternal diabetes mellitus induces oxidative stress, which causes endoplasmic reticulum stress by aggravating unfolding protein response events in the endoplasmic reticulum. Oxidative stress and endoplasmic reticulum stress induce phosphorylation of the Thr-845 that is present on the activation loop of apoptosis signal-regulating kinase 1, thereby activating apoptosis signal-regulating kinase 1. Apoptosis signal-regulating kinase 1 activation then leads to the phosphorylation of c-Jun N-terminal kinase 1/2, which activates several transcription factors. These transcription factors ultimately induce endothelial progenitor cell apoptosis and senescence.

ASK1 , apoptosis signal-regulating kinase 1; ER , endoplasmic reticulum; JNK1/2 , c-Jun N-terminal kinase.

Dong. The role of yolk sac in diabetic embryopathy. Am J Obstet Gynecol 2016 .

ASK1-mediated apoptosis is involved in the pathogenesis of several oxidative stress-related diseases such as brain ischemia, ischemic heart disease, and Alzheimer’s disease. ASK1 activation leads to apoptosis via the JNK or the p38MAP kinase pathways. ASK1 is activated by phosphorylation of Thr-845 in its activation loop, and ASK1 is required for reactive oxygen species (ROS) and ER stress-induced JNK activation and apoptosis. Recently, it has been shown that high glucose-induced activation of ASK1 mediates hyperglycemia-induced endothelial cell senescence. We have demonstrated that ASK1 is activated in diabetic yolk sac vasculopathy and that ASK1 deletion morphologically ameliorates diabetic yolk sac vasculopathy. This indicates that ASK1 mediates maternal diabetes mellitus–induced endothelial progenitor apoptosis or senescence by JNK1/2 and that activation of the ASK1-JNK1/2 pathway leads to vasculopathy ( Figure 2 ).

Altered nitric oxide and nitric oxide synthase (NOS) in yolk sac vasculopathy

Nitric oxide is a small multifunctional gaseous molecule that acts as a vasoactive modulator, signaling molecule, and free radical in mammalian systems. Nitric oxide is synthesized from oxidation of L-arginine by 3 distinct nitric oxide synthases (NOS): neuronal (nNOS), endothelial (eNOS), and inducible (iNOS), using the cofactors, reduced nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, and tetrahydrobiopterin. nNOS and eNOS are expressed constitutively at low levels. nNOS generates very high concentrations of nitric oxide only when induced. Nitric oxide has been shown to be involved in cell differentiation, proliferation, and apoptosis; the effect of nitric oxide is both physiologically essential and cytotoxic. Upon generation, nitric oxide freely diffuses through the cell membrane into the extracellular space and subsequently modifies protein thiols or cysteine residues. In addition, nitric oxide induces a variety of biologic responses by interacting with free radicals. Nitric oxide interacts with several signaling pathways to mediate these responses, including MAPK, Janus kinase, and JNK pathways and reactive oxygen, depending on signaling pathways.

During blood island formation in diabetic pregnancies, the endoderm produces nitric oxide, which inhibits NOS. Inhibition of NOS, L-N ^G -monomethyl arginine citrate leads to developmental arrest at the primary plexus stage and ultimately vasculopathy. Administration of an nitric oxide donor reverses these adverse effects on yolk sac vasculature. Additionally, it has been reported that nitric oxide derived from iNOS plays a detrimental role in human disease. Moreover, iNOS and eNOS are expressed during early embryonic vasculogenesis, and the alteration of nitric oxide expression induces yolk sac vasculopathy. Hyperglycemia increases iNOS protein expression and activity through ASK1. The increase of iNOS leads to over-production of nitric oxide that causes DNA damage, ER stress, nuclear factor kappa B, and respiratory inhibition that may play a vital role on embryonic malformation ( Figure 3 ).

Overproduction of nitric oxide mediates maternal diabetes mellitus–induced yolk sac vasculopathy

Maternal diabetes mellitus–induced oxidative stress activates apoptosis signal-regulating kinase 1. The phosphorylation of apoptosis signal-regulating kinase 1stimulates induced nitric oxide synthase gene expression, which generates very high concentrations of nitric oxide. The detrimental role of nitric oxide derived from induced nitric oxide synthase includes DNA damage, endoplasmic reticulum stress, nuclear factor kappa B inhibition, and respiratory inhibition, all of which contribute to yolk sac vasculopathy.

ASK1 , apoptosis signal-regulating kinase 1; iNOS , induced nitric oxide synthase; NF-kB , nuclear factor kappa B; NO , nitric oxide.

Dong. The role of yolk sac in diabetic embryopathy. Am J Obstet Gynecol 2016 .

The protective effect of the ASK1 inhibitor thioredoxin-1 in yolk sac vasculopathy

Thioredoxin-1 (Trx) is a 12-kd protein with a redox-active dithiol in the active site (-Cys-Gly-Pro-Cys-) and constitutes a major thiol-reducing system. Trx is a potent antioxidant and reduces ROS through interactions with its redox-active center, which protects cells from stress-induced damage through antioxidative, antiapoptotic, and antiinflammatory effects. Trx shows an antiapoptotic function by inhibiting cell death signals, activating survival signaling pathways, or scavenging ROS. Diabetic yolk sac vasculopathy is an oxidative stress and apoptotic disease process. Therefore, Trx is able to reduce diabetic yolk sac vasculopathy via its antioxidative and antiapoptotic functions ( Figure 4 ).

Thioredoxin-1 reduces diabetic yolk sac vasculopathy by scavenging reactive oxygen species

Reduced thioredoxin-1 is a potent antioxidant that decreases reactive oxygen species levels through the function of its redox-active center. Thioredoxin-1 ultimately protects cells from stress-induced damage by antioxidative, antiapoptosis, and antiinflammation processes. Maternal diabetes mellitus–induced oxidative stress disturbs the redox balance of thioredoxin-1, leading to a disproportionate increase in oxidized thioredoxin-1. High levels of oxidized thioredoxin-1 are associated with several cardiovascular diseases, which include atherosclerosis, vascular injuries, ischemia reperfusion injury, hypertension, and yolk sac vasculopathy. The therapeutic strategy for maternal diabetes mellitus–associated embryopathy may be through induction or overexpression, and deoxidation of thioredoxin.

ROS , reactive oxygen species; Trx , thioredoxin-1.

Dong. The role of yolk sac in diabetic embryopathy. Am J Obstet Gynecol 2016 .

Trx is expressed ubiquitously in mammalian cells, and its expression is essential for early differentiation and morphogenesis of the mouse embryo. Genetic deletion of Trx leads to an early embryonic lethal phenotype. Trx-deficient embryos die shortly after implantation, and the conceptuses are resorbed before gastrulation. Preimplantation Trx-null embryos are placed in culture, the inner mass cells of the homozygous embryos fail to proliferate. This indicates that proper levels of Trx are essential for normal embryogenesis. Trx levels are reduced in embryonic tissues that exposed to diabetes mellitus, which implies that Trx reduction is involved in the pathogenesis of diabetic emrbyopathy.

Trx is expressed ubiquitously in endothelial cells and protects them from ROS-induced apoptosis. Trx is active in the vessel wall and functions either as an important endogenous antioxidant or interacts directly with signaling molecules to influence cell growth, apoptosis, and inflammation. Recent evidence implicates that Trx is involved in cardiovascular diseases that are associated with oxidative stress, such as atherosclerosis, vascular injuries, ischemia reperfusion injury, and hypertension. In vivo studies have shown a protective role of Trx in different cardiovascular diseases. Thus, Trx is considered an important target for therapeutic intervention of cardiovascular disorders.

It has also been reported that Trx stimulates angiogenesis via induction of angiogenic factors. For example, hyperglycemia-induced yolk sac vasculopathy in mice can be ameliorated by treatment with exogenous human Trx recombinant protein. Based on the profound beneficial effects of Trx on vascular functions and diabetic vasculopathy, induction or overexpression and deoxidation of Trx is able to reverse hyperglycemia-induced yolk sac vasculopathy ( Figure 4 ).

Therapeutic implications of targeting the yolk sac

The leading intervention strategy that currently is applied to prevent diabetic embryopathy is rigorous glycemic control with lifestyle modifications and various antidiabetic agents, such as insulin, and other therapies, such as antihypertensives, as needed. Unfortunately, continuous euglycemic control is difficult to achieve and maintain; even transient exposure to hyperglycemia causes embryonic malformation.

Our group has shown that fatty acid supplements have some beneficial effects on the outcome of diabetic pregnancies. We analyzed the fatty acid composition in major lipid groups of the yolk sac in rats and found that maternal diabetes mellitus induces quantitative and qualitative abnormalities in major lipid groups of the yolk sac. This implies that the teratogenic mechanism of diabetic embryopathy may be related to a deficiency in essential fatty acids in the yolk sac. In addition, we used dietary supplementation of arachidonic acid and myo -inositol, in vitro and in vivo, and showed that these substrates can reduce the incidence of diabetes mellitus–related malformation in offspring.

Previous work also has indicated that arachidonic acid prevents hyperglycemia-associated yolk sac damage and embryopathy. When rodent conceptuses were cultured in normal, arachidonic acid-supplemented normal, and arachidonic acid-supplemented hyperglycemic rat serum, the addition of 20 mg/ml of arachidonic acid prevented open neural tubes, increased the number of lysosome-like structures in the visceral endodermal yolk sac cells, advanced neuropil formation in the neuroepithelium, significantly reduced ER, and decreased size and number of lipid droplets in embryos that were cultured under high glucose conditions.

Dietary myo -inositol supplements also appear to decrease the incidence of neural tube defects in offspring of diabetic dams significantly. The results of a previous study showed that dietary therapy successfully restored myo -inositol levels in the yolk sac and reduced malformation. These therapies hold promise for use as a dietary prophylaxis against diabetic embryopathy in humans.