Objective
Maternal hyperglycemia increases the risk of congenital malformations. Epigallocatechin-3-gallate (EGCG), a natural antioxidant purified from green tea, inhibits oxidative stress signaling. We propose that EGCG prevents hyperglycemia-induced malformation via inhibition of oxidative stress signaling. The objective of this study is to examine the effect of EGCG on hyperglycemia-induced adverse effects during embryonic development.
Study Design
Day-9 rat conceptuses were cultured under euglycemic (150 mg/dL glucose) and hyperglycemic (300 mg/dL glucose) conditions in the presence or absence of 1 or 10 μmol/L of EGCG.
Results
Both 1 and 10 μmol/L of EGCG significantly ameliorated hyperglycemia-induced embryonic vasculopathy and malformations. Hyperglycemia inactivated protein kinase B (Akt) by reducing phosphorylated Akt levels. EGCG reversed the inhibitory effect of hyperglycemia on Akt activation. EGCG also prevented hyperglycemia-reduced phosphorylated Forkhead transcription factor 3a levels.
Conclusion
EGCG prevented hyperglycemia-induced embryopathy through inhibition of Forkhead transcription factor 3a activation. This may have been mediated via the activation of Akt. These findings offer the potential for a possible pharmacological prophylaxis for hyperglycemia-induced embryonic malformations.
Congenital malformations in children of diabetic women are significant clinical problems and the recent increase in diabetes makes this pregnancy complication a continuing issue. Because glycemic control in diabetic women is difficult to control and maintain, malformation rates of children of diabetic women are at least 6 times higher than those in nondiabetic women. Therefore, therapeutic interventions in addition to insulin are needed for diabetes-associated adverse pregnancy outcomes. Mechanistic studies are essential steps for the development of accessible, convenient, and effective prevention strategies. Published data support the conclusion that congenital malformations during maternal hyperglycemia are the result of a disruption in the balance between intracellular reactive oxygen species and endogenous antioxidant capacities. Thus, embryonic malformations under hyperglycemic conditions are the results of oxidative stress. Animal studies with dietary supplement of antioxidants such as multivitamins effectively prevent diabetic embryopathy. However, human studies with conventional antioxidants do not result in any beneficial effects for human diseases associated with oxidative stress.
Prevention strategies for diabetic embryopathy must meet 2 important criteria: high effectiveness and lack of teratogenicity. Epigallocatechin-3-gallate (EGCG), a polyphenolic component of green tea, has high antioxidant capacities and does not possess any teratogenic effects even at very high doses. In addition to its antioxidant properties, EGCG is also a cell signaling modulator, suppressing proapoptotic signaling and promoting prosurvival signaling in nontumor cells. This is highly relevant to diabetic embryopathy. Hyperglycemia-induced oxidative stress activates proapoptotic signaling pathways and suppresses prosurvival pathways that lead to apoptosis in target embryonic cells. Oxidative stress-triggered apoptosis results in embryonic malformations. Since EGCG has both antioxidant and antioxidative stress signaling properties, it is reasoned that EGCG may be more effective in prevention of hyperglycemia-induced malformations than conventional antioxidants.
Our previous studies demonstrated a causative role of the proapoptotic c-Jun-N-terminal kinase (JNK)1/2 in the induction of diabetic embryopathy. Forkhead transcription factors, Forkhead transcription factor (Foxo)1, Foxo3a, and Foxo4, are downstream effectors of JNK1/2. Foxo factors are also oxidative stress mediators and apoptosis inducers. Foxo3a is enhanced whereas the activity of prosurvival signaling intermediate Akt (protein kinase B) is decreased. Akt and Foxo signaling are closely interrelated with the negative regulation of Foxo activities by Akt. Because EGCG is a potent cell signaling modulator that favors cell survival, we will evaluate the effect of EGCG in the present study on altered Foxo3a and Akt signals by hyperglycemia.
The well-established whole-conceptus culture system is used to examine the effect of EGCG on hyperglycemia-induced adverse effects on embryonic development during period of organogenesis. The whole-conceptus culture system coupled with hyperglycemia faithfully mimics maternal diabetes-induced adverse pregnancy outcomes, avoids the maternal influences, and enables us to directly assess the protective effects of EGCG on conceptuses exposed to hyperglycemia. Embryonic vasculature is the first system to develop during embryonic development. Hyperglycemia induces embryonic vasculopathy resulting in either embryonic malformations or lethality. Using quantitative measurements, we have found that EGCG significantly reduces both hyperglycemia-induced embryonic vasculopathy and malformations, blocks hyperglycemia-induced Foxo3a activation, and reverses the inhibition of Akt by hyperglycemia. EGCG may be a possible therapeutic candidate for diabetic embryopathy.
Materials and Methods
Reagents
Sprague-Dawley rats were purchased (Charles River Laboratories, Wilmington, MA). EGCG was purchased (Sigma, St Louis, MO) and dissolved in water.
Whole-conceptus culture
The procedures for animal use were approved by the University of Maryland School of Medicine Institutional Animal Care and Use Committee. Sprague-Dawley rats were paired overnight. Vaginal smears were examined the next morning, and the presence of spermatozoa in the examination indicated embryonic day (E) 0. Rat conceptuses at E9 were dissected out of the uteri in phosphate-buffered saline (Invitrogen, La Jolla, CA). The parietal yolk sac was removed using a pair of fine forceps and the visceral yolk sac was left intact. Conceptuses (4/bottle) were cultured in 4 mL of rat serum at 38°C in 30 rpm rotation in the roller bottle system. For the initial 24 hours of culture, the bottles were gassed with 5% O 2 /5% CO 2 /90% N 2 . For the following 12 hours, the bottles were gassed with 20% O 2 /5% CO 2 /75% N 2 , and in the last 12 hours, 40% O 2 /5% CO 2 /75% N 2 were applied. Conceptuses were cultured under euglycemic (150 mg/dL glucose) and hyperglycemic (500 mg/dL glucose) conditions or in the presence and absence of 1 or 10 mmol/L of EGCG. After 48-hour culture, conceptuses were harvested for morphological examinations of yolk sac vasculature and embryonic malformations.
Morphologic assessment of the yolk sac vasculature and embryonic malformations
Conceptuses were examined under a stereomicroscope (MZ16F; Leica, Bannockburn, IL) to assess yolk sac vasculature defect, then yolk sacs and embryos were separated and embryonic malformations were identified. Images of embryos with or without yolk sacs were captured by a digital camera (DFC420 5 MPix; Leica) with software and processed with Photoshop CS2 (Adobe Systems Inc., Seattle, WA).
Yolk sac vasculatures were morphologically scored based on visible maldevelopment as previously described. A morphological score of 4 indicated full development of the E11-like yolk sac vasculature with an arborizing interconnecting vascular network composed of arteries, veins, and capillaries exhibiting blood flow. A score of 3 represented only a minor defect of the yolk sac vasculature with fewer blood vessels than that of the yolk sac vasculature with score 4. A score of 2 indicated an arrest of the yolk sac vasculature development at the primary capillary plexus stage resulting in few yolk sac vessels. A score of 1 indicated a major defect of the yolk sac vasculature displaying an ecstatic vascular plexus with no signs of arborization and large, nonfused blood islands toward the ectoplacental cone. A score of 0 represented a completely devoid of the yolk sac vasculature with no visible or scattered blood islands. Normal embryos were classified as exhibiting correct body flexure, completely closed neural tube, and no evidence of other malformations. Malformed embryos were classified as showing evidence of failed closure of the neural tube or neural tube defect and other malformations including cardiac abnormalities, incorrect body curvature, reverse tail flexion, and microcephaly.
Western blotting
Western blotting was performed as described by Yang at el. Briefly, after 48-hour culture, embryos were collected and were sonicated in 80-μL of ice-cold lysis buffer (20 mmol/L Tris-hydrochloride pH 7.5, 150 mmol/L sodium chloride, 1 mmol/L ethylenediaminetetraacetic acid, 10 mmol/L sodium fluoride, 2 mmol/L sodium orthovanadate, 1 mmol/L phenylmethanesulfonylfluoride, and 1% Triton 100) containing a protease inhibitor cocktail (Sigma). Equal amounts of protein (50 μg) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany). Membranes were incubated with primary antibodies against phosphor-threonine 32-Foxo3a (Cell Signaling, Beverly, MA) or phospho-threonine 473-Akt (Cell Signaling), followed by peroxidase-conjugated secondary antibodies. Membranes were exposed to goat antirabbit (Jackson ImmunoResearch Laboratories, West Grove, PA) secondary antibodies. To ensure that equivalent amounts of protein were loaded among samples, membranes were stripped and probed with a mouse antibody against β-actin (Abcam, Cambridge, MA). Signals were detected using an Amersham ECL Advance Detection Kit (GE Healthcare, Piscataway, NJ) and chemiluminescence emitted from the bands was directly captured using a UVP Bioimage EC3 system (UVP, Upland, CA). Densitometric analysis of chemiluminescence signals were performed by software (VisionWorks LS; UVP). Images of representative immunoblots were arranged using Photoshop and PowerPoint (Microsoft Corp., Redmond, WA) software. All experiments were repeated 3 times with the use of independently prepared tissue lysates.
Statistics
Data are presented as means ± SE. One-way analysis of variance was performed using SigmaStat 3.5 software (Systat Software, Inc., San Jose, CA). In analysis of variance analysis, Tukey test was used to estimate the significance of the results. Statistical significance was accepted at P < .05. Significant difference between groups in malformation incidences was analyzed by χ 2 test.
Results
EGCG significantly ameliorates hyperglycemia-induced embryonic vasculopathy
Defective embryonic vasculatures result in either embryonic lethality or congenital malformations. A morphological score system has been developed to quantitatively assess the defects of yolk sac vasculatures. Conceptuses cultured under hyperglycemic conditions had lower morphological scores than those cultured under euglycemic conditions ( Figure 1 , A). EGCG at concentrations 1 and 10 μmol/L did not have any adverse effects on embryonic vasculature development because vasculature morphological scores in EGCG groups did not differ from those in the euglycemic control group ( Figure 1 , B). EGCG treatment at 1 and 10 μmol/L significantly reversed the reduction of vasculature morphological scores by hyperglycemia ( Figure 1 , B). Conceptuses cultured under euglycemia or under hyperglycemia with EGCG had fully developed vasculature networks after 48-hour culture (a, b, c, e, and f of Figure 1 , A). In contrast, conceptuses cultured under hyperglycemia alone were devoid of vessels (a of Figure 1 , A) or had variety of defects ranging from arrest at the blood island stage to the presence of few blood vessels.
EGCG effectively reduces hyperglycemia-induced embryonic malformations
Hyperglycemia-induced oxidative stress and resultant apoptosis are responsible for hyperglycemia-induced malformations. EGCG is an antioxidant and is able to inhibit proapoptotic signals. To determine if EGCG reduces hyperglycemia-induced malformations, conceptuses cultured under hyperglycemia (300 mg/dL glucose) were treated with 1 or 10 μmol/L of EGCG. As previously reported, hyperglycemia resulted in a 50% embryonic malformation rate ( Table ). Conceptuses cultured under euglycemia (150 mg/dL) had a low embryonic malformation rate (7.14%) ( Table ). No embryonic malformations were observed in conceptuses cultured under euglycemia plus 1 or 10 μmol/L of EGCG ( Table ), suggesting that not only does EGCG have no teratogenic effects, but it also supports normal embryonic development. With 1- or 10-μmol/L EGCG treatment, hyperglycemia-induced malformations were completely prevented ( Table ). After 48-hour culture, E9 embryos in the euglycemia group, and the hyperglycemia plus 1 or 10 μmol/L of EGCG groups, developed to normal E11-like structures with well-closed neural tube and correct body curvature ( Figure 2 , A, C, and D). In contrast, embryos in the hyperglycemia group exhibited neural tube defects with failed neural tube closure and incorrect body curvature ( Figure 2 , B). These results provide strong in vitro evidence in support of EGCG as a possible therapeutic candidate for hyperglycemia-induced embryonic anomalies.
