Objective
Maternal diabetes adversely impacts embryonic vasculogenesis, which results in embryonic vasculopathy. The purpose of our study is to determine whether hypoxia inducible factor (HIF)-1α plays a role in diabetic embryonic vasculopathy.
Study Design
Levels of HIF-1α were determined in mouse conceptuses. Conceptuses on day 7 of pregnancy were cultured under euglycemic (150 mg/dL glucose) and hyperglycemic (300 mg/dL) conditions with or without AdCA5, or in the presence or absence of 2.0 μg/mL human recombinant thioredoxin, an endogenous antioxidant protein. AdCA5 is an adenovirus encoding a constitutively active form of HIF-1α.
Results
Maternal diabetes significantly reduced HIF-1α protein expression. The administration of 1 μL (1 × 10 7 infectious units/mL) per 1 mL culture medium AdCA5 completely reversed hyperglycemia-reduced vasculature morphological scores and vascular endothelial growth factor expression. Thioredoxin treatment reversed hyperglycemia-reduced HIF-1α levels.
Conclusion
We conclude that reduced HIF-1α plays a critical role in the induction of diabetic embryonic vasculopathy, and that oxidative stress is implicated in hyperglycemia-induced HIF-1α reduction.
Congenital malformations occur in up to 10% of babies born to diabetic women, and the recent rise in diabetes makes this pregnancy complication an extraordinarily important issue. The embryonic vasculature is the first system to be developed and is the most vulnerable to the in utero environmental conditions. Hyperglycemia has been shown to be associated with embryonic vasculopathy, which leads to embryonic lethality or malformations. The mechanism underlying maternal diabetes-induced embryonic vasculopathy is elusive. Most studies have focused on the mechanisms of maternal diabetes-induced malformations and have determined that congenital malformations during maternal hyperglycemia are the result of a disruption in the balance between intracellular reactive oxygen species and endogenous antioxidant capacities. This oxidative-stress hypothesis is also applicable to maternal diabetes-induced embryonic vasculopathy because our recent study has found that a natural antioxidant purified from green tea, epigallocatechin-3-gallate, is effective in amelioration of hyperglycemia-induced embryonic vasculopathy in vitro. Although oxidative stress mediates the negative impact on embryonic vasculogenesis, it is not clear how maternal diabetes and consequent oxidative stress adversely regulate vascular factors leading to embryonic vasculopathy.
Hypoxia inducible factor (HIF)-1 is a key transcriptional factor for hypoxic regulation of embryonic vascular development. HIF-1α is the oxygen-sensitive subunit of HIF-1. Regulation of HIF-1 activity is critically dependent on the degradation of the HIF-1α subunit in normoxia. HIF-1 acts as a master regulator of angiogenesis by controlling the expression of multiple angiogenic growth factors, including vascular endothelial growth factor (VEGF). Mice lacking HIF-1 activity due to HIF-1α null mutation develop extensive vascular defects similar to those observed in diabetic embryonic vasculopathy, including inadequate vessel formation and aberrant vascular remodeling. Increased or reduced HIF-1α protein levels contribute to the pathogenesis of several diabetic complications. Because the stability of HIF-1α is pivotal to its functions in embryonic vasculogenesis, we chose to assess both HIF-1α gene expression and protein levels in diabetic embryonic vasculopathy.
Embryonic vasculogenesis begins in the yolk sac (extraembryonic membrane) prior to vasculogenesis in the embryo. In addition, development of the embryonic cardiovascular system and yolk sac vasculature are regulated by the same group of angiogenic and survival factors via common mechanisms. Thus, previous studies in hyperglycemia-induced embryonic vasculopathy have specifically focused on the yolk sac vasculature because it has provided a highly reliable experimental system. The yolk sac is an extraembryonic membrane derived from the same progenitor cells that produce the embryo, and it plays an important role in supporting development of embryos. Adverse effects of hyperglycemia have been documented in the yolk sac of maternal diabetic animal models and in vitro cultured rodent embryos. Under hyperglycemic conditions, vasculogenesis of the blood vessels in the yolk sac is disrupted, and the cellular structures in the vessels are altered.
We have morphologically characterized the various adverse effects of hyperglycemia on yolk sac vasculature development by arbitrarily assigning morphological scores to individual vasculatures. We have successfully used this established morphological score system in our studies to quantify the adverse effect of hyperglycemia on yolk sac vasculature development.
In the present study, we used this yolk sac morphological system to test the hypothesis that hyperglycemia reduces HIF-1α expression, and blockade of HIF-1α reduction ameliorates diabetic embryonic vasculopathy. By using in vivo and in vitro models with a constitutively active form of HIF-1α, AdCA5, we have demonstrated that hyperglycemia reduces HIF-1α protein expression, but not messenger RNA (mRNA) expression, and reversal of HIF-1α reduction by AdCA5 reduces diabetic embryonic vasculopathy.
Materials and Methods
Animals and reagents
C57BL/6J mice (median body weight 22 g) were purchased from Jackson Laboratory (Bar Harbor, ME). Streptozotocin from Sigma (St Louis, MO) was dissolved in sterile 0.1 mol/L citrate buffer (pH 4.5). Sustained-release insulin pellets were purchased from LinShen Canada Inc. (Toronto, Canada). Adenoviruses expressing the LacZ (AdLacZ) and the constitutive active form of HIF-1α (AdCA5) were provided by Dr Gregg L. Semenza at the Johns Hopkins University School of Medicine, Baltimore, MD. Human recombinant thioredoxin (Trx) was purchased from EMD Chemicals (San Diego, CA).
Mouse models of diabetic embryopathy
The procedures for animal use were approved by the Institutional Animal Care and Use Committee of University of Maryland School of Medicine. Eight-week-old C57BL/6J mice were intravenously injected daily with 75 mg/kg streptozotocin over 3 days to induce diabetes. Once a level of hyperglycemia indicative of diabetes (≥250 mg/dL) was achieved, insulin pellets were subcutaneously implanted in these diabetic mice to restore euglycemia prior to mating. The mice were then mated with male mice of the same respective genotype. On day 5 of pregnancy (E5), insulin pellets were removed to permit frank hyperglycemia (>250 mg/dL glucose level), so the developing conceptuses would be exposed to a hyperglycemic environment during organogenesis (E7–11). Nondiabetic female mice with vehicle injections and sham operation of insulin pellet implants served as nondiabetic controls. On E7 and E8, mice were euthanized, and conceptuses were dissected out of the uteri for analysis.
Whole-conceptus culture
C57BL/6J mice were paired overnight. The next morning was designated E0 if a vaginal plug was present. Mouse conceptuses at E7 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 rat serum at 38°C in 30 rpm rotation in the roller bottle system. The culture bottles were gassed with 5% oxygen/5% carbon dioxide/90% nitrogen. Conceptuses were cultured under euglycemic (150 mg/dL glucose, a value close to the blood glucose level of nondiabetic mice) and hyperglycemic (300 mg/dL glucose) conditions in the presence or absence of 0.5 μL or 1 μL (1 × 10 7 infectious units/mL) adenoviral AdCA5 per 1 mL culture medium, or in the presence or absence of 2.0 μg/mL human recombinant Trx.
Morphologic assessment of the yolk sac vasculature
Conceptuses were examined under a stereomicroscope (MZ16F; Leica Microsystems Inc, Bannockburn, IL) to assess yolk sac vasculature defects. Images of conceptuses were captured by a DFC420 5 MPix digital camera with software (Leica, Wetzlar, Germany) and processed with Adobe Photoshop CS2 (Adobe Systems Incorporated, San Jose, CA).
Yolk sac vasculatures were morphologically scored based on visible maldevelopment as previously described. Briefly, a morphological score of 4 indicated a 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 a score of 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 yolk sac completely devoid of blood vessels with no visible or scattered blood islands.
Embryonic malformations were not examined because at early embryonic stages (E7-E9), structural malformations were not manifested. Our previously studies have extensively described the malformations in embryos of diabetic mice or cultured embryos exposed to hyperglycemia. Especially, at E11, about 25% of embryos from diabetic mice exhibited neural tube defects. Our ongoing studies are testing the hypothesis that the early molecular changes involving embryonic vasculopathy play causative roles in the induction of embryonic malformations in late development stages.
Real-time polymerase chain reaction
Total RNA was isolated from E7 and E8 conceptuses retrieved from nondiabetic or diabetic mice using an RNeasy Mini Kit (Qiagen, Valencia, CA). Real-time polymerase chain reaction (PCR) for HIF-1α and β-actin were performed using ABI TaqMan Gene Expression Assays (assay ID: Mm00468875_m1 and Mm00607939_s1, respectively; Applied Biosystems, Foster City, CA). Briefly, RNA was reverse transcribed by using the high-capacity cDNA archive kit (Applied Biosystems). Real-time PCR and subsequent calculations were performed by a 7700 ABI PRISM sequence detector system (Applied Biosystems), which detected the signal emitted from fluorogenic probes during PCR.
Western blotting
Western blotting was performed as described by Yang at el. Briefly, embryonic samples were sonicated in 80 μL ice-cold lysis buffer (20 mmol/L Tris-HCl pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 10 mmol/L NaF, 2 mmol/L Na orthovanadate, 1 mmol/L PMSF, and 1% Triton 100) containing a protease inhibitor cocktail (Sigma). Equal amounts of protein (50 μg) were resolved by SDS-PAGE and transferred onto a nitrocellulose membrane (Schleicher and Schuell, Keene, NH). Membranes were incubated for 18 hours at 4°C with the following primary antibodies at 1:1000-1:2000 dilutions in 5% nonfat milk: rabbit anti-HIF-1α (Sigma) or rabbit anti-VEGF (catalog no. ab9953; Abcam, Cambridge, MA). Membranes were exposed to goat antirabbit, antimouse (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). 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 VisionWorks LS software (UVP). Images of representative immunoblots were arranged using Adobe Photoshop and Microsoft PowerPoint software (Richmond, CA). 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 were performed using SigmaStat 3.5 software (Systat software, San Jose, CA). In analysis of variance, Tukey test was used to estimate the significance of the results. Statistical significance was accepted at P < .05.
Results
Hyperglycemia reduces HIF-1α protein expression with no effects on its mRNA levels
To determine if maternal diabetes affects HIF-1α expression, E7 and E8 conceptuses from nondiabetic and diabetic mice were used for detection of HIF-1α protein and mRNA. Maternal diabetes significantly reduced HIF-1α protein in both E7 and E8 conceptuses ( Figure 1 , A). HIF-1α protein levels in E7 nondiabetic conceptuses were higher than those E8 nondiabetic conceptuses, consistence with the notion that the degree of embryonic hypoxia is gradually reduced with the advance of pregnancy. To determine if alternation of HIF-1α protein is due to the change of its mRNA, HIF-1α mRNA levels were determined in E7 and E8 conceptuses. Maternal diabetes did not alter HIF-1α mRNA levels ( Figure 1 , B). The levels of HIF-1α (mRNA and protein) reflect the levels in the whole conceptuses including the embryo and the yolk sac.
Adenoviral gene delivery is effective in cultured conceptuses with no teratogenic effects, and AdCA5 ameliorates hyperglycemia-induced vasculopathy
To establish the novel approach, adenoviral gene delivery to cultured conceptuses, E7 conceptuses were cultured in the presence or absence of 1 μL (1×10 7 infectious units/mL) adenoviral vector encoding GFP (AD-CMV-GFP, adenovirus expresses enhanced green fluorescent protein under the control of a CMV promoter.) (Vector BioLabs, Philadelphia, PA) per 1 mL culture medium. After 48-hour culture, whole-mount yolk sacs were examined for green fluorescence-labeled cells under a fluorescence microscope. Virtually, all yolk sac cells exposed to AD-CMV-GFP expressed GFP ( Figure 2 , A). To determine if adenovirus infections have any teratogenic effects, embryonic malformations were examined in conceptuses exposed to AD-CMV-GFP. No malformations were observed in embryos exposed to adenoviruses and embryonic development in conceptuses exposed to AD-CMV-GFP was comparable to that in unexposed conceptuses ( Figure 2 , B and C). We also did experiments with 3 or 4 μL (1 × 10 7 IFU/mL) AD-CMV-GFP and did not observe any teratogenic effects on embryonic development.
