Hyperglycemia impairs cytotrophoblast function via stress signaling

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OBSTETRICS

Hyperglycemia impairs cytotrophoblast function via stress signaling Chase R. Cawyer, MD, MBA; Darijana Horvat, BS; Dean Leonard; Steven R. Allen, MD; Richard O. Jones, MD; David C. Zawieja, PhD; Thomas J. Kuehl, PhD; Mohammad N. Uddin, PhD OBJECTIVE: Diabetes mellitus is a risk factor for preeclampsia.

Cytotrophoblast (CTB) invasion is facilitated from the conversion of plasminogen to plasmin by urokinase plasminogen activator (uPA), regulated by plasminogen activator inhibitor 1 (PAI-1), and may be inhibited in preeclampsia. This study assessed signaling mechanisms of hyperglycemia-induced CTB dysfunction. STUDY DESIGN: Human CTBs were treated with 45, 135, 225, 495, or

945 mg/dL glucose for 48 hours. Some cells were pretreated with a p38 inhibitor (SB203580) or a peroxisome proliferator-activated receptor-gamma (PPAR-g) ligand (rosiglitazone). Expression of uPA, PAI-1, and PPAR-g levels and p38 mitogen-activated protein kinase phosphorylation were measured by Western blot in cell lysates. Messenger ribonucleic acid of uPA and PAI-1 was measured by quantitative polymerase chain reaction. Levels of interleukin-6, angiogenic (vascular endothelial growth factor [VEGF], placenta growth factor [PlGF]) and antiangiogenic factors (soluble fms-like tyrosine kinase-1 [sFlt-1], soluble endoglin [sEng]) were measured in the media by enzyme-linked immunosorbent assay kits. Statistical

comparisons were performed using analysis of variance with a Duncan’s post-hoc test. RESULTS: Both uPA and PAI-1 protein and messenger ribonucleic acid

were down-regulated (P < .05) in CTBs treated with 135 mg/dL glucose or greater compared with basal (45 mg/dL). The sEng, sFlt-1, and interleukin-6 were up-regulated, whereas the VEGF and PlGF were down-regulated by 135 mg/dL glucose or greater. p38 phosphorylation and PPAR-g were up-regulated (P < .05) in hyperglycemiatreated CTBs. The SB203580 or rosiglitazone pretreatment showed an attenuation of glucose-induced down-regulation of uPA and PAI-1. CONCLUSION: Hyperglycemia disrupts the invasive profile of CTB by

decreasing uPA and PAI-1 expression; down-regulating VEGF and PlGF; and up-regulating sEng, sFlt-1, and interleukin-6. Attenuation of CTB dysfunction by SB203580 or rosiglitazone pretreatment suggests the involvement of stress signaling. Key words: angiogenesis, cell invasion, cytotrophoblast cells, hyperglycemia

Cite this article as: Cawyer CR, Horvat D, Leonard D, et al. Hyperglycemia impairs cytotrophoblast function via stress signaling. Am J Obstet Gynecol 2014;211:541.e1-8.

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reeclampsia (preE) is a complex syndrome that is produced by various pathophysiological triggers and mechanisms affecting 3-8% of pregnancies worldwide.1-3 PreE has a higher incidence in women with diabetes mellitus than in the nondiabetic population (about 1 in 5 vs about 1 in 20, respectively).4,5 Although

the pathogenesis of preE is largely unknown, many investigations have focused on the incompleteness of placental invasion of cytotrophoblast (CTB) cells6-9; these same explorations have yet to be studied with reference to diabetes. CTB cells are essential to the development of a successful pregnancy. During

From the Departments of Obstetrics and Gynecology (Drs Cawyer, Allen, Jones, Kuehl, and Uddin and Ms Horvat) and Medical Physiology (Dr Zawieja), Scott and White Healthcare/Texas A&M Health Science Center College of Medicine, Temple, and Prehealth Studies (Mr Leonard), Baylor University, Waco, TX. Received Feb. 24, 2014; revised March 28, 2014; accepted April 28, 2014. This study was supported by the Scott, Sherwood, and Brindley Foundation, Department of Obstetrics and Gynecology (M.N.U.), and the Noble Centennial Endowment for Research in Obstetrics and Gynecology (T.J.K.), Scott and White Healthcare, Temple, TX. The authors report no conflict of interest. Presented in oral format at the 34th annual meeting of the Society for Maternal-Fetal Medicine, New Orleans, LA, Feb. 3-8, 2014. Reprints: Mohammad Nasir Uddin, PhD, Scott & White Hospital (MS-01-E316A), 2401 South 31st St., Temple, TX 76508. [email protected] 0002-9378/$36.00 ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajog.2014.04.033

the first trimester, CTB cells take on an invasive character as extravillous CTB cells invade and reduce resistance within the spiral arteries, thus allowing for optimal maternal blood transport to the placenta.10 Disrupted invasion by CTB cells hinders arterial remodeling, causing shallow placentation, which predisposes to preeclampsia.11 Extracellular matrix digestion via proteinase activation of the plasmin pathway facilitates CTB invasiveness in the endometrium.12 Urokinase plasminogen activator (uPA) acts independently of fibrin and is involved in the regulation of cell adhesion and migration of trophoblastic cells.13,14 The uPA messenger ribonucleic acid (mRNA) and immunoreactivity have been detected in rhesus monkey CTB cells and in first- and thirdtrimester human decidual cells.15 The expression of plasminogen activator inhibitor-1 (PAI-1) in CTB cells is integral in establishing a functional

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FIGURE 1

Western blots from CTB cell lysates treated with glucose in vitro

markers mentioned in previous text in relation to cardiotonic steroids.22,39 Consistent with other studies,1,40 this demonstrated that marinobufagenin inhibits CTB proliferation, migration, and invasion.22,41 Using similar methodology, we evaluated how hyperglycemiainduced stress signaling has an impact on the invasive phenotype and angiogenic balance of CTB cells to help better understand the relationship between diabetes and preeclampsia.

M ATERIALS

Representative Western blots from CTB cell lysates treated with various concentrations of glucose in vitro. Some cultures were pretreated with either a p38 inhibitor or rosiglitazone prior to exposure to various concentrations of glucose. The number of replicate plates for each glucose series is included with the test agent. CTB, cytotrophoblast. Cawyer. Glucose on CTB cells. Am J Obstet Gynecol 2014.

maternal-fetal interface15 and has been demonstrated to be affected by the p38 pathway.16 CTB proliferation, differentiation, invasiveness, and apoptosis are all influenced directly by mitogen-activated protein kinase (MAPK)17-22 and indirectly by interleukin-6 (IL-6)23,24 during times of cellular stress. Peroxisome proliferator-activated receptor gamma (PPAR-g), a subtype of the ligandactivated transcription factor superfamily, stimulates villous trophoblastic differentiation and proliferation25-28 and has been evaluated as a potential therapeutic target in preeclampsia.29 In normal pregnant serum, PPAR-g activators up-regulate the expression and activity of the PPAR-g receptor.30,31 Inversely, PPAR-g agonists, such as rosiglitazone, cause inhibition of extravillous CTB cell invasion through competitive

binding with the retinoid X receptor-a heterodimers in vitro.32 Revascularization at the placental interface also involves multiple regulatory pathways of angiogenic and antiangiogenic factors. Vascular endothelial growth factor (VEGF) promotes the syncytialization and proliferation of extravillious trophoblast.33-35 Placental growth factor (PlGF), a member of the VEGF subfamily, is expressed by CTB cells and is fundamental for angiogenesis.36 Antiangiogenic markers include soluble fms-like tyrosine kinase1 (sFlt-1), a VEGF receptor antagonist), and the capillary tube inhibitor soluble endoglin (sEng).37 In previous studies, our research team demonstrated a correlation between levels of a marinobufagenin, a urinary marker elevated in patients with preeclampsia,38 and an angiogenic imbalance of the

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CTB cell culture The human extravillous CTB cell line Sw.71 utilized in these studies was derived from first-trimester chorionic villus tissue and was kindly provided by Dr Gil G. Mor (Yale University School of Medicine, New Haven, CT). These cells are well characterized and share many characteristics with isolated primary cells, including the expression of cytokeratin-7, human leukocyte antigen class I antigen, human leukocyte antigenG, BC-1, CD9, human chorionic gonadotropin, and human placental lactogen. Sw.71 cells were cultured in Dulbecco’s modified Eagle’s medium/F-12 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 10 mM N-2-hydroxyethylpipera-zineN’-2-ethane sulfonic acid, 0.1 mM minimal essential medium nonessential amino acids, 1 mM sodium pyruvate, and 100 U/mL penicillin/streptomycin. Cells were incubated at 37 C, 6% CO2, 5% O2, and 99% humidity (Isotemp CO2 incubator; Fisher, Waltham, MA), with no exposure to hypoxic conditions. Effect of hyperglycemia on CTB cells CTB cells were seeded on 6-well plates. Prior to treatment, cells were incubated in serum-free media for 24 hours. Cells were treated with 45 (basal), 135, 225, 495, or 945 mg/dL of glucose (Sigma, St. Louis, MO) for 72 hours. Each plate had 2 wells assigned to the basal condition and 1 well assigned to each of the 4 increased glucose levels. Thus, 1 plate constituted 1 replicate for a series of glucose exposures. Some cell suspensions were pretreated with 10 mM of a p38 inhibitor (SB203580) or 10 mM of a

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ajog.org PPAR-g ligand (rosiglitazone) for 3 hours prior to seeding into plates with the glucose treatments.

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FIGURE 2

p38 MAPK phosphorylation relative to p38a/b levels and PPAR-g relative to b-actin with CTB cells exposed to glucose

Antibodies and primers Primary antibodies The following antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA): uPA (H-140), catalog no. sc-14019, concentration, 1:500; PAI-1 (H-135), catalog no. sc-8979, concentration, 1:500; PPAR-g (H-100), catalog no. sc-7196, concentration, 1:500; p-p38 (Tyr 182)-R, catalog no. sc-7975-R, concentration, 1:500; p38a/b (H-147), catalog no. sc-7149, concentration, 1:500; and b-actin (C4), catalog no. sc-47778, concentration, 1:10,000. Secondary antibodies The following antibodies were purchased from Cell Signaling Technology, Inc (Danvers, MA): antimouse immunoglobulin G, horseradish peroxidase-linked antibody, catalog no. 7076, concentration, 1:2000; and antirabbit immunoglobulin G, horseradish peroxidase-linked antibody, catalog no. 7074, concentration, 1:5000. Primers The following primers were purchased from QIAGEN (Valencia, CA): RT2 quantitative polymerase chain reaction (qPCR) primer assay for human plasminogen activator, urokinase, also known as uPA, catalog no. PPH00796C200, Entrez Gene identification 5328; RT2 qPCR primer assay for human serpin peptidase inhibitor, clade E, also known as PAI-1, catalog no. PPH00215F200, Entrez Gene identification 5054; RT2 qPCR primer assay for human glyceraldehyde-3-phosphate dehydrogenase, catalog no. PPH00150F-200, Entrez Gene identification 2597; and RT2 qPCR primer assay for human b-actin (actin, beta), also known as b-actin, catalog no. PPH00073G-200, Entrez Gene identification 60. Western blot for uPA, PAI-1, and PPAR-g expression and p38 MAPK phosphorylation After treatment for 72 hours, the media were removed from cells and a lysis

Plot of p38 MAPK phosphorylation relative to p38a/b levels in 10 replicate experiments and PPAR-g relative to b-actin in 5 replicated experiments with CTB cells exposed to various levels of glucose. The p38 MAPK phosphorylation is increased (P < .05) in CTB cells treated with 495 mg/dL or greater of glucose compared with basal (45 mg/dL), whereas PPAR-g expression is increased (P < .05) in 135 mg/dL or greater glucose. The means with different letters differ (P < .05). CTB, cytotrophoblast; MAPK, mitogen-activated protein kinase; PPAR-g, peroxisome proliferator-activated receptor-gamma. Cawyer. Glucose on CTB cells. Am J Obstet Gynecol 2014.

buffer (Cell Signaling Technology) containing 50 mM Tris at pH 7.4, 50 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate, 0.3 mM Naorthovanadate, 50 mM NaF, 1 mM dichlorodiphenyl-trichloroethane, 10 mg/mL leupeptin, and 5 mg/mL aprotinin was added to the cells. Cells were scraped and put into tubes. Protein concentrations were determined by a bicinchoninic assay reagent (Pierce, Rockford, IL). An equal amount of protein in each sample was separated using NuPAGE Novex 4-12% Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes. Membranes were blocked in 5% milk and probed with uPA (Santa Cruz Biotechnology), PAI-1 (Santa Cruz Biotechnology), PPAR-g (Santa Cruz Biotechnology), phospho-p38 (Santa Cruz Biotechnology), p38a/b

(Santa Cruz Biotechnology), and b-actin (Santa Cruz Biotechnology) antibodies. After incubation with the corresponding secondary antibody, proteins were visualized with a chemiluminescence detection system (Pierce). The intensity of the bands was determined using ImageQuant LAS 4000 (GE Healthcare, Life Sciences, Indianapolis, IN). The expression of uPA, PAI-1, PPARg, and p38 MAPK phosphorylation protein was quantified by a densitometry analysis using Image J software (National Institutes of Health, Bethesda, MD) in which the target protein (uPA, PAI-1, or PPAR-g) is normalized to a structural protein (b-actin) to control between groups and ensures correction for the amount of total protein on the membrane (phospho-p38 is normalized to total p38).


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FIGURE 3

uPA protein and mRNA expression for CTB cells exposed to hyperglycemia and pretreatment with a p38 inhibitor or rosiglitazone

Results are normalized to values with basal glucose (45 mg/dL) and those values labeled with an asterisk differ (P < .05) from basal values. The means with SE for 8 replicates are shown for uPA protein in control conditions with 8 for p38 inhibitor exposure and 4 for rosiglitazone treatment. qPCR was performed with 4 replicates for each treatment. Glucose levels of 225 mg/dL or greater decreased (P < .05) the uPA protein expression, whereas levels of 135 mg/dL or greater decreased (P < .05) uPA mRNA levels. Pretreatment with p38 inhibitor or rosiglitazone attenuated these changes. GADPH, glyceraldehyde 3-phosphate dehydrogenase; mRNA, messenger ribonucleic acid; qPCR, quantitative polymerase chain reaction; uPA, urokinase plasminogen activator. Cawyer. Glucose on CTB cells. Am J Obstet Gynecol 2014.

These replicative glucose exposure experiments were performed 8 times with uPA without inhibitor pretreatment, 8 times with pretreatment using p38 inhibitor, and 4 times with pretreatment using rosiglitazone. For PAI-1 there were 14 replicates without inhibitors, 6 with pretreatment using p38 inhibitor, and 4 with pretreatment with rosiglitazone. The glucose series was repeated 5 times for PPAR-g and 10 times for p38 MAPK phosphorylation. Sample sizes for these replicates are shown in Figure 1 with representative immunoblots.

Quantitative PCR for uPA and PAI-1 After the treatment, the media were removed from cells, and a lysis/binding

solution from the Ambion RNAqueous4PCR kit (Invitrogen) was added to the cells. Cells were scraped and put into tubes. An equal amount of 64% ethanol was added to the tubes and mixed. The mixed solution was added to a filter and centrifuged into another tube. The filter was then washed with wash solution #1 from the kit and then wash solution #2/ #3 from the kit. Preheated elution solution from the kit was added to the filter to elute the ribonucleic acid (RNA) into tubes. Then 10 mL of 10 times deoxyribonuclease (DNAse) I buffer and 1 mL of DNAse I from the kit was added to the RNA tubes. Samples were incubated in a heat block at 37 C for 30 minutes. Then 11 mL of the DNAse inactivation reagent


from the kit was added to the samples and mixed for 2 minutes and then centrifuged. A Nanodrop (Thermo Fisher Scientific, Wilmington, DE) was used to get the concentration of RNA in each sample. Then the sample tubes were heated for 3 minutes at 75 C and put on ice. In a new tube, 2 mL of oligo(deoxythymidine) from the Ambion RETROscript firststrand synthesis kit (Invitrogen) was added to 10 mL of RNA. Then 2 mL of 10 times reverse transcriptase buffer from the kit, 4 mL of deoxynucleotide triphosphate mix from the kit, 1 mL ribonuclease inhibitor from the kit, and 1 mL Molony-murine leukemia virus reverse transcriptase from the kit were added to the tubes for a total volume of 20 mL. Tubes were mixed gently and spun down. Tubes were placed in a Perkin Elmer GeneAmp 9600 PCR thermal cycler and set to incubate at 42-44 C for 1 hour and then incubate at 92 C for 10 minutes to inactivate the reverse transcriptase. The Nanodrop was then used to get the concentration of complementary deoxyribonucleic acid in each sample. For the real-time polymerase chain reaction (PCR), the primers used were uPA, serpin peptidase inhibitor, clade E (PAI-1), glyceraldehyde-3-phosphate dehydrogenase, and ACTB (b-actin), all purchased from QIAGEN. Also used was iTaq SYBR Green Supermix with ROX (Bio-Rad Laboratories, Hercules, CA). Each well of the PCR plate contained 1 mL of the complementary deoxyribonucleic acid, 1 mL of the primer, 12.5 mL of SYBR Green, and 10.5 mL of water. Quantitative PCR was performed on a Bio-Rad iCycler iQ5 (Bio-Rad Laboratories), using a 2-step cycling program. Results were analyzer using LinRegPCR software from Dr J. M. Ruijter (The Heart Failure Research Center, Amsterdam, The Netherlands). The replicated glucose exposure experiments were performed 4 times with uPA mRNA without inhibitor pretreatment and 4 times with each inhibitor pretreatment. Similarly, qPCR for PAI-1 mRNA was replicated 4 times without inhibitors and 4 times using each inhibitor pretreatment.

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ajog.org Enzyme-linked immunosorbent assay for a sEng, sFlt-1, VEGF 165, PlGF, and IL-6 After the treatment, the media removed from the cells were place in tubes. Levels of antiangiogenic (sEng, sFlt-1) and angiogenic (VEGF 165, PlGF) factors as well as the level of inflammatory cytokine IL-6 were measured in the culture media by the commercially available enzyme-linked immunosorbent assay (ELISA) kits from R&D Systems (Minneapolis, MN). For sENG, a human endoglin/CD105 quantikine ELISA kit was used. For sFLT-1, a human soluble VEGF R1/Flt-1 quantikine ELISA kit was used. For VEGF 165, a human VEGF quantikine ELISA kit was used. For PlGF, a human PlGF quantikine ELISA kit was used. For IL-6, a human IL-6 quantikine ELISA kit was used. The replicated glucose exposure experiments were performed 4 times for each factor. Statistical methods Data are expressed as mean SE. Statistical significance is assessed by analysis of variance and a Duncan’s post-hoc test for differences between glucose effects and inhibitor treatments, with P < .05 taken as significant. While interpreting the grafts, asterisks and different letters will signify a statistical significance.

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FIGURE 4

PAI-1 protein and mRNA expression for CTB cells exposed to hyperglycemia and pretreatment with a p38 inhibitor or rosiglitazone

Plots of PAI-1 protein expression relative to b-actin and PAI-1 mRNA expression relative to b-actin mRNA for cytotrophoblast cells responding in vitro to hyperglycemia and treatment with p38 inhibitor or rosiglitazone. Results are normalized to values with basal glucose (45 mg/dL) and those values labeled with an asterisk differ (P < .05) from basal values. The means with SE for 14 replicates are shown for PAI-1 protein in control conditions with 6 for p38 inhibitor exposure and 4 for rosiglitazone treatment. qPCR was performed with 4 replicates for each treatment. Glucose levels of 225 mg/dL or greater decreased (P < .05) PAI-1 protein expression and PAI-1 mRNA. Pretreatment with the p38 inhibitor or rosiglitazone primarily attenuated the mRNA levels with minimal impact of protein expression during the 48 hours of culture in comparison. CTB, cytotrophoblast; IL-6, interleukin-6; mRNA, messenger ribonucleic acid; PAI-1, plasminogen activator inhibitor 1; qPCR, quantitative polymerase chain. Cawyer. Glucose on CTB cells. Am J Obstet Gynecol 2014.

R ESULTS Hyperglycemia up-regulated p38 MAPK phosphorylation and PPAR-g expression Figure 2 demonstrates that the ratio of phosphorylated p38 MAPK to the nonphosphorylated p38a/b was significantly (P < .05) up-regulated in all the CTB cell cultures treated with 495 mg/dL or more of glucose compared with basal (45 mg/dL). Intermediate levels of glucose exposure, beginning at more than 225 mg/dL, produced intermediate changes that were not different from values at either exposure to 45 mg/dL or 495 mg/dL. In addition, Figure 2 shows that PPAR-g expression was significantly up-regulated in 135 mg/dL or more

glucose-treated CTB cells compared with basal (45 mg/dL).

Hyperglycemia down-regulated uPA protein and mRNA expression Graphs in Figure 3 show that uPA protein and mRNA levels in CTB cells are altered by increasing concentrations of glucose in the culture medium. Glucose levels of 225 mg/dL or greater decreased uPA protein expression compared with basal glucose at 45 mg/dL. However, this decrease was seen at a lower level of 135 mg/dL in uPA mRNA levels. Pretreatment with either a p38 inhibitor or rosiglitazone attenuated these changes.

Hyperglycemia down-regulated PAI-1 protein and mRNA expression As shown in Figure 4, there is no real statistical difference in hyperglycemia’s effect of PAI-1 protein expression at any glucose level. At glucose levels of 225 mg/dL or greater, there was a decrease (P < .05) in PAI-1 mRNA levels (P < .05) in comparison with basal glucose (45 mg/dL). Pretreatment with p38 inhibitor or rosiglitazone primarily attenuated mRNA levels with a minimal impact of protein expression during the 72 hour culture in comparison. PAI-1 is significantly increased only in the presence of the p38 inhibitor.


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ajog.org C OMMENT

FIGURE 5

Angiogenic factors, antiangiogenic factors, and IL-6 measured by ELISA from culture CTB cells exposed to hyperglycemia

Bar graphs of angiogenic factors, antiangiogenic factors, and IL-6 as measured by an ELISA in media from culture CTB cells exposed to hyperglycemia. Values are normalized to basal glucose (45 mg/dL). Mean values are labeled with letters. Those means without the same letter differ (P < .05) using Duncan’s post-hoc test. Means with SE are derived from 4 replicate experiments. Exposure to glucose levels of 135 mg/dL or greater increases sEng, sFlt-1, and IL-6 secretion and decreases VEGF 165 and PlGF secretion into the media. CTB, cytotrophoblast; ELISA, enzyme-linked immunosorbent assay; IL-6, interleukin-6; PlGF, placental growth factor; sEng, soluble endoglin; sFlt-1, soluble fms-like tyrosine kinase-1; uPA, urokinase plasminogen activator; VEGF, vascular endothelial growth factor. Cawyer. Glucose on CTB cells. Am J Obstet Gynecol 2014.

Hypergycemia up-regulated sEng, sFlt-1, and IL-6 expression and down-regulated VEGF and PlGF expression As shown in Figure 5, the antiangiogenic factors (sENG and sFLT-1) were significantly (P < .05) up-regulated in 135 mg/ dL or greater glucose-treated CTB cells

in comparison with basal (45 mg/dL). The angiogenic factors (VEGF165 and PlGF) were significantly (P < .05) downregulated in the presence of 135 mg/dL or greater glucose compared with basal. The level of IL-6 was higher in the presence of glucose 135 mg/dL or greater compared with basal (45 mg/dL) conditions.


As confirmed by multiple other investigators, preeclampsia is commonly associated with inadequate endovascular cytotrophoblast function.8,9 Postulating supraphysiological hyperglycemia as an environmental stressor, we demonstrate an induction of PPAR-g expression that is consistent with that reported by Suwaki et al42 yet differing with Jawerbaum et al.43 These results also are consistent with those reported by Zhou et al,40 who illustrated that hyperglycemia activates multiple MAPK pathways through an up-regulation of the active phosphorylated p38 pathway, thus confirming our suspicion of glucose as a stress signal. Multiple investigators have demonstrated that preE is commonly associated with inadequate endovascular cytotrophoblast invasion.44,45 We herein present evidence of hyperglycemia-induced disruption of the plasmin pathway through a dose effect down-regulation for uPA and PAI-1. Differing from what is seen in preeclampsia,46 mRNA PAI-1 is not up-regulated but is down-regulated at glucose concentrations greater than 225 mg/dL compared with basal. Furthermore, these effects of hyperglycemia are attenuated by the p38 inhibitor or rosiglitazone pretreatment. It is interesting to note that at all concentrations less than 945 mg/dL, there was a trend, although not statistically significant, toward the up-regulation of PAI-1 mRNA in the presence of the p38 inhibitor and rosiglitazone. Although an invasion assay was not performed to determine a direct concentration effect, it does not dispute the fact that glucose disrupts the invasive characteristics. With reference to the angiogenic profile, the hyperglycemia-induced changes of the CTB cells are consistent with those seen in preeclampsia.1,37,46 Excess glucose down-regulated the angiogenic factors (VEGF and PlGF), whereas it upregulated the antiangiogenic factors (sEng and sFlt-1) and the inflammatory marker, IL-6. Although we report a dose-effect impact of hyperglycemia beginning at the upper range of physiological values

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FIGURE 6

Possible mechanisms of hyperglycemia induced CTBs dysfunction

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relationship between diabetes mellitus and placental pathology. These initial studies allow us to build investigator interest and institutional teams to work toward disease prevention.

REFERENCES

Hyperglycemia impedes CTBs by induction of stress signaling (p38 MAPK and PPAR-g) followed by the inhibition of uPA and PAI-1, leading to CTBs invasion problems, and the up-regulation of sFlt-1, sEng, and IL-6 and the down-regulation of VEGF and PlGF, leading to antiangiogenic milieu. These changes cumulatively contribute to a final common pathway of placental dysfunction, thus development of the preE syndrome. CTB, cytotrophoblast; IL-6, interleukin-6; MAPK, mitogen-activated protein kinase; PAI-1, plasminogen activator inhibitor 1; PlGF, placental growth factor; PPAR-g, peroxisome proliferator-activated receptor-gamma; preE, preeclampsia; sEng, soluble endoglin; sFlt1, soluble fms-like tyrosine kinase-1; uPA, urokinase plasminogen activator; VEGF, vascular endothelial growth factor. Cawyer. Glucose on CTB cells. Am J Obstet Gynecol 2014.

to supraphysiological values upon multiple markers in this study, our study is limited in that we do not specify at which concentrations these alterations begin to occur. Although demonstrating significant stress cell signaling often occurring at 135 mg/dL compared with 45 mg/dL, it is unclear whether these cell signals are a part of the natural physiological processes in CTB cells and begin at normal fasting glucose levels or at the beginning of an elevated glucose state. Now that we have reported this finding, additional studies with more glucose levels are justified to clarify this issue. However, in considering the clinical application of this study, we can appreciate that there are a number of cellular physiological changes that can be induced by glucose levels within a range that is likely to be more common in

patients with hyperglycemia in pregnancy. These changes suggest that the CTB cells respond to elevations in glucose as a stress signal. Placental pathology in diabetic pregnancies demonstrates impaired trophoblastic differentiation seen as biochemical and structural abnormalities.47 Such alterations, including inadequate cytotrophoblast invasion, may mediate the association between maternal diabetes and preeclampsia. As summarized in Figure 6, all of these changes through the induction of stress signaling appear to contribute to a final common pathway that leads to abnormal placentation and the possible development of preeclampsia. Being able to understand the pathophysiological development of cytotrophoblast cells in the presence of excess glucose will give us a better understanding of the

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