BIOLOGY OF REPRODUCTION (2014) 90(2):42, 1–9 Published online before print 18 December 2013. DOI 10.1095/biolreprod.113.115592

Maternal and Fetoplacental Hypoxia Do Not Alter Circulating Angiogenic Growth Effectors During Human Pregnancy1 Stacy Zamudio,2,3,4,5 Marcus Borges,6 Lourdes Echalar,7 Olga Kovalenko,6 Enrique Vargas,7 Tatiana Torricos,8 Abdulla Al Khan,3,4 Manuel Alvarez,3,4 and Nicholas P. Illsley3,4,9 3

Department of Obstetrics and Gynecology, Division of Maternal Fetal Medicine and Surgery, Hackensack University Medical Center, Hackensack, New Jersey 4 Center for Abnormal Placentation, Hackensack University Medical Center, Hackensack, New Jersey 5 Department of Preventive Medicine and Community Health, Rutgers-New Jersey Medical School, Newark, New Jersey 6 Department of Obstetrics Gynecology and Women’s Health, Rutgers-New Jersey Medical School, Newark, New Jersey 7 Instituto Boliviano de Biologı´a de Altura, Universidad de San Andreas Mayor, La Paz, Bolivia 8 Hospital Hernandez Vera, Villa Primer de Mayo, Santa Cruz, Bolivia 9 Deparment of Pharmacology and Physiology, Rutgers-New Jersey Medical School, Newark, New Jersey INTRODUCTION

One causal model of preeclampsia (PE) postulates that placental hypoxia alters the production of angiogenic growth effectors (AGEs), causing an imbalance leading to maternal endothelial cell dysfunction. We tested this model using the natural experiment of high-altitude (HA) residence. We hypothesized that in HA pregnancies 1) circulating soluble fms-like tyrosine kinase 1 (sFlt-1) is increased and placental growth factor (PlGF) decreased, and 2) AGE concentrations correlate with measures of hypoxia. A cross-sectional study of healthy pregnancies at low altitude (LA) (400 m) versus HA (3600 m) compared normal (n ¼ 80 at HA, n ¼ 90 at LA) and PE pregnancies (n ¼ 20 PE at HA, n ¼ 19 PE at LA). Blood was collected using standard serum separation and, in parallel, by a method designed to inhibit platelet activation. AGEs were measured by enzyme-linked immunosorbent assays. AGEs did not differ between altitudes in normal or PE pregnancies. AGE concentrations were unrelated to measures of maternal or fetal hypoxia. PlGF was lower and sFlt-1 higher in PE, but overlapped considerably with the range observed in normal samples. PlGF correlated with placental mass in both normal and PE pregnancies. The contribution of peripheral cells to the values measured for AGEs was similar at LA and HA, but was greater in PE than in normotensive women. Hypoxia, across a wide physiological range in pregnancy, does not alter levels of circulating AGEs in otherwise normal pregnancies. Peripheral cell release of AGEs with the hemostasis characteristic of standard blood collection is highly variable and contributes to a doubling of the amount of sFlt-1 measured in PE as compared to normal pregnancies.

Attention has focused on circulating angiogenic growth effectors and their binding proteins (AGEs) in pregnant women as being causally related to the human-specific pregnancy syndrome known as preeclampsia (PE) [1–4]. AGEs implicated in PE are free vascular endothelial growth factor (VEGF), free placental growth factor (PlGF), and their binding protein, soluble fms-like tyrosine kinase 1 (sFlt-1). Endoglin [5] and, more recently, soluble endoglin [6] as well as splice variants of sFlt-1 [7, 8] have also emerged as potential biomarkers and mediators of endothelial cell dysfunction. Soluble Flt-1 can limit growth factor-stimulated transactivation by sequestering VEGF and PlGF or by forming inactive heterodimers with the transmembrane receptors Flk and Flt-1 [9]. In PE, the causal argument is that binding of free VEGF and PlGF by excess sFlt-1 inhibits their beneficial actions on the vascular endothelium, permitting the systemic endothelial cell damage that is thought to precipitate PE symptoms [10]. This is reflected in the circulation of some preeclamptic women by high levels of sFlt-1 and low levels of PlGF and free VEGF. Why sFlt-1 is elevated in preeclamptic pregnancy is not completely understood. However oxygen tension is a major regulator of the expression of AGEs in the placenta [11–17]. Specifically, sFlt-1 increases with lowered oxygen tension, and PlGF and VEGF decrease with higher oxygen tension [18–20]. All three AGEs are subject to regulation by the nuclear transcription factor hypoxia-inducible factor-1 (HIF-1), which is elevated in PE [16, 21]. This has led to the hypothesis that placental hypoxia causes excess production of sFlt-1 and thus decreases maternal circulating free PlGF and free VEGF in preeclamptic pregnancies. We tested this hypothesis using the natural experiment afforded by human residence at high altitude (HA), in which maternal arterial PO2 is diminished by ;30% and fetal PO2 by ;10%, and there is clear evidence of maternal, placental, and fetal hypoxia [16, 22–24]. The incidence of PE is increased by 2- to 4-fold at HA, supporting the idea that hypoxia is contributory [25, 26]. Many of the normal physiological changes of pregnancy are altered at HA so that values are intermediate between normal pregnancy at sea level and PE [27]. Levels of HIF-1alpha are doubled in HA placentae, similar to what is reported in PE [21, 22]. Given that we have shown that the mother, fetus, and placenta are hypoxic relative to sea-level controls, we would expect higher levels of

altitude, methods of blood collection, monocytes, PBMCs, platelet inhibition, platelets, PlGF, preeclampsia, sFlt-1, VEGF 1 Supported by NIH HD042737, TW007444, and NSF BCS-0309142 (all to S.Z.), and HD046982 (to N.I.). 2 Correspondence: Stacy Zamudio, Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine and Surgery, Hackensack University Medical Center, 30 Prospect Ave. WCP-4E-90G, Hackensack, NJ 07601. E-mail: [email protected]

Received: 4 November 2013. First decision: 5 December 2013. Accepted: 11 December 2013. Ó 2014 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363

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ABSTRACT

ZAMUDIO ET AL. blood collection, and immediately centrifuged for 10 min at 2000 3 g at 48C to collect plasma. Samples were flash frozen in liquid nitrogen and stored at 808C until analysis. None of the samples tested for this report had been previously thawed. We chose the CTAD method based on reports indicating that failure to control for clotting time invalidates the use of VEGF as an indicator of disease states [33–35]. The other AGEs have not, to our knowledge, been tested with this method before our recent examination of the effects of hemostasis on AGE concentrations in maternal and fetal cord blood in normal LA pregnancies [28].

hypoxia-stimulated AGEs and lower levels of those unresponsive to hypoxia. We tested two specific hypotheses. First, we predicted that we would find increased sFlt-1 and decreased PlGF in HA pregnancy. Second, we hypothesized that AGE levels would correlate with maternal and/or fetal oxygenation. We did not measure free VEGF because we have shown that detectable levels are an artifact of blood collection technique in both the maternal and fetal circulation. In the same study, we demonstrated that 83%, 29%, and 20% of maternal circulating levels of VEGF, PlGF, and sFlt-1, respectively, were attributable to peripheral cell release/secretion and highly variable from individual to individual [28]. In the fetal circulation, peripheral cell contribution to AGEs using standard blood collection methods was 98% of free VEGF and 59% of sFlt-1, with a significant increase in the number of samples with nondetectable levels [28]. Therefore, in this study we also tested to what extent altered concentrations in AGEs, if present, may be due to secretion/release by other cell types within the circulation. AGEs are known to be produced/stored/bound/ released by peripheral cells in the circulation, and it is possible that chronically lowered maternal PO2 may alter these processes [29–31].

Assays All the enzyme-linked immunosorbent assay kits were purchased from or donated by R&D Systems (sVEGF R1/Flt-1 [DVR-100] and free PlGF [DPG00]; Minneapolis, MN). Curve-fitting (linear or polynomial) was used for generation of standard curves and subsequent calculation of the unknown (sample) values. Not all the assays could be done on all the subjects because never-thawed samples were not available for some women. Sample sizes for each assay are given in the figure legends.

Validation Studies

MATERIALS AND METHODS Research Design, Subjects, and Sites The present study was part of a larger, cross-sectional study evaluating the effects of altitude and genetic ancestry on uterine and umbilical blood flows, O2 delivery/consumption, placental function, and pregnancy outcome [23, 24]. Genetic ancestry was assessed using 133 single nucleotide polymorphisms that permit individual estimates of biogeographic ancestry, for example, a participant might be 80% Native American, 12% European, 4% sub-Saharan African, and 4% East Asian. For the variables we report here, biogeographic ancestry had no impact. Therefore, we compared all the women at low altitude (LA) (400 m, n ¼ 90) with all HA pregnancies (3600 m, n ¼ 80). An additional 39 pregnancies complicated by PE, 20 at HA and 19 at LA, were compared with their respective altitude controls. All the participants gave written, informed consent as approved by the Bolivian National Bioethics Committee, the local Institutional Review Boards, and our U.S. Institutional Review Board. Inclusion criteria were good health (absence of chronic conditions that predispose to PE, e.g., hypertension, renal disease, or obesity), conception, gestation, and delivery at the altitude of study. Women were excluded for drug, alcohol, or tobacco use, or a positive oral glucose tolerance test. All the women remained at their altitude of residence for their entire pregnancy. Preeclampsia was defined using National Institutes of Health consensus guidelines [32]. Hypertension was defined as 140 mmHg systolic or 90 mmHg diastolic on two or more occasions in previously normotensive pregnancies in the third trimester. Proteinuria was present where readings of 2 þ on dipstick and/or 0.3 g protein in a 24-h urine collection were present in the absence of urinary tract infection. The sample size for PE does not permit division into the most informative hierarchy, that is, early onset PE and lateonset PE with versus without intrauterine growth restriction. The data are therefore divided into early onset of symptoms (,34 wk) and delivery at ,35.9 wk gestational age, and late onset cases in which symptoms and delivery occurred later than 36 wk.

PlGF The detection limit is reported as 7.0 pg/ml. A 4-fold dilution was determined as optimal and used for all the samples. All the samples had detectable levels of PlGF, and none were ,7 pg/ml.

Soluble Flt-1 The reported detection limits ranged from 1.5–13.3 pg/ml, with a mean of 3.5 pg/ml. We measured matched serum and CTAD plasma samples from the normal and preeclamptic mothers. A 20-fold dilution was required in the maternal samples. All the serum samples had detectable levels of sFlt-1. In the maternal preeclamptic samples, 20- to 100-fold dilutions were required to obtain values within the range of the standards.

Statistical Analysis None of the AGEs passed the D’Agostino and Pearson normality test. Thus, for the matched serum versus CTAD samples, a Wilcoxon signed rank test was used to determine whether the conditions of blood collection influenced the results. The Mann-Whitney U-test was used to compare between altitudes. Data are presented as box and whiskers or scatter plots. In box plots, the median is indicated by the bar, the 25th and 75th centiles by the box, and the maximum and minimum by whiskers. In the text and legends, brackets [ ] indicate the interquartile range. Maternal and fetal demographic and clinical data are reported as the mean 6 SEM. Comparison between the normal and preeclamptic subject groups was analyzed by the Kruskal-Wallis test followed by Dunn multiple comparison test. Categorical data was analyzed by chi square. Regression analyses were used to compare the relationship between AGE concentrations, gestational age, birth or placental weight, and maternal or fetal measures of oxygenation. Values were considered significant where P was  0.01; the P value was reduced (Bonferroni correction) because the AGEs in normal women were tested three times, by altitude, compared with PE and by serum versus CTAD blood collection procedures.

Blood Collection and Measures of Oxygenation One to ten days prior to elective cesarean delivery (mean 6 SEM, 3 6 1 days), mothers completed a health screen and medical history. An arterialized blood sample (warmed hand vein) was drawn for measurement of blood gases and oxygen content as previously described [23]. Upon admission for cesarean delivery, a 15 ml maternal venous blood sample was distributed into serum separator vacutainers at room temperature and into ice-cold vacutainers containing sodium citrate, theophylline, adenosine, and dipyridamole (CTAD) as previously described [28]. CTAD is added to prevent platelet activation and release of platelet-derived factors into the plasma, but CTAD likely prevents activation of other peripheral cells as well. No supplemental oxygen was used prior to maternal blood sampling. Blood samples were allowed to clot for 30 min at room temperature; serum was then separated by centrifugation (2000 3 g for 10 min). The CTAD tubes were prechilled, placed on ice before and after

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Validation studies have been reported in detail elsewhere [28]. Briefly, to ensure that the CTAD reagent was not interfering with the enzyme-linked immunosorbent assay, the linearity of the standard curves with versus without CTAD was evaluated. The curves were overlapping and linear with an r2  0.98. In addition, to ensure that a CTAD/plasma reaction would not alter the results, we examined a heparin-treated plasma sample (no CTAD) with a known concentration of the AGE of interest. When diluted for analysis, one aliquot was treated with standard diluent and the other with diluent plus 15 ll CTAD reagent. The values obtained did not differ between the CTAD treated versus untreated samples. For each assay, spike and recovery experiments and serial dilutions were used to test the dynamic range of the assay, to determine the extent to which experimental samples required dilution to fall within the standard curves, and to assure full recovery at high and low ends of the range of values. Linearity tests in the PlGF assay yielded an r2 of 0.96 and of 0.98 for sFlt-1. The intraassay coefficient of variation (CV 6 SD) was calculated for the duplicate samples within each plate and averaged across all the plates. Interassay variation was calculated using the pooled samples mentioned above, in triplicate on every plate. For PlGF, the intraassay CV was 3.6% 6 8.9%, and for sFlt-1, the CV was 3.2% 6 6.1%. The interassay CV was 9% 6 8% for PlGF and 15% 6 10% for sFlt-1.

ANGIOGENIC GROWTH EFFECTORS IN HYPOXIC PREGNANCY

Soluble Flt-1

Normotensive Participants

Concentrations of sFlt-1 did not differ between LA and HA (Fig. 2A). Collection of blood into CTAD reduced sFlt-1 concentrations in healthy pregnant women by 22% 6 3% and 23% 6 3% at 400 and 3600 m, respectively (Fig. 2B). Values obtained with CTAD also did not differ between altitudes. The range of variation was large, from 0% to 69% decrease at LA and from 0% to 83% decrease at HA. One subject at LA and three at HA had a decline in sFlt-1 values with CTAD, but the difference was less than 7%. There was no correlation with gestational age or placental or birth weight. The initial, serumderived values for sFlt-1 were unrelated to the percent decrease with CTAD (r2 ¼ 0.00, P ¼ 0.89 at LA; r2 ¼ 0.01, P ¼ 0.72 at HA). In summary, the results for these AGEs show that in both plasma and serum samples, none differ by altitude in healthy, normal pregnancies despite lowered maternal and fetal PO2. Moreover, the results from CTAD plasma samples show that a third of the circulating free PlGF and nearly a quarter of sFlt-1 in serum samples collected under standard clinical conditions are derived from other cells in the maternal circulation. The degree to which hemostasis alters these AGE concentrations is highly variable from individual to individual and unrelated to having either high or low values, precluding any form of standard correction for hemostatic effects.

Table 1 shows the characteristics of the healthy women and their neonates. Mothers were generally similar in their demographic characteristics, but birth weight was lower in the HA pregnancies despite similarity in gestational age. Adjustment for variation in maternal height, body mass index, weight gain with pregnancy, and parity and for neonatal gestational age and fetal sex did not appreciably change the birth weights (Table 1). In short, demographic factors do not explain the altitude-associated decrement in birth weight, which is accompanied by reduced body length and abdominal circumference but preserved head circumference. Mothers at 3600 m relative to 400 m had lower PO2 and blood oxygen saturation (SaO2, P , 0.0001). Arterial PO2 was 54 6 1 mmHg (range 46–72) whilst at 400 m, PaO2 was 92 6 1 mmHg (range 84–115). Arterialized blood is not as reliable as data obtained by direct arterial puncture for measurement of PO2; saturation (SaO2) is more reliable and ranged from 94% to 99% at LA and from 85% to 94% at HA. The fetuses also had lower umbilical venous PO2 (27 6 1 mmHg at 3600 m, range, 15–41, vs. 31 6 1 mmHg at 400 m, range, 9–46, P , 0.01), but not SaO2 [24]. PlGF PlGF concentrations were similar at 400 and 3600 m and were not decreased at HA as predicted (Fig. 1A). The values obtained from CTAD samples also did not differ by altitude (Fig. 1B). However CTAD treatment decreased PlGF by onethird relative to the paired serum samples (Fig. 1B). The decrement ranged from 11% to 67% at LA and from 13% to 78% at HA. The initial, serum-derived values for PlGF were unrelated to the percent decrease with CTAD (r2 ¼ 0.02, P ¼ 0.58 at LA; r2 ¼ 0.00, P ¼ 0.94 at HA), that is, whether initial PlGF values were high or low did not appear to influence the change observed with CTAD. Values were unrelated to gestational age or birth weight. However at both altitudes, maternal circulating PlGF concentrations were positively correlated with placental weight (Fig. 1C, r2 at each altitude ¼ 0.17, P , 0.001).

Women with Preeclampsia The characteristics of and clinical data pertinent to the mothers and infants in the preeclamptic samples are given in Table 2. As would be expected, these subjects were younger, virtually all were primiparous, had lower gestational age at delivery, and had lower birth and placental weights than their normotensive counterparts. The median and interquartile ranges for PlGF and sFlt-1 are also given in Table 2 for the early and late-onset groups; however, because there was no significant difference between these groups and given the small sample size, the data were consolidated for the figures and statistical analyses. Maternal oxygen tension in preeclamptics was 93 6 3 mmHg (range: 80–114) at 400 m and 59 6 3 mmHg (range: 44–75) at 3600 m; similar to those obtained in

TABLE 1. Maternal and neonatal characteristics. Altitude Characteristics

400 m (n ¼ 90)

3600 m (n ¼ 80)

Maternal Age (yr) Gravidity Parity Proportion primiparous (%) Height (cm) Nonpregnant weight (kg) Nonpregnant body mass index (kg/m2) Weight gain with pregnancy (kg)

28 6 1 2.7 6 0.2 1.3 6 0.2 50% 159 6 1 60 6 1 24.0 6 0.4 11.5 6 0.5

32 6 1 2.7 6 0.2 1.3 6 0.1 30% 157 6 1 60 6 1 24.3 6 0.5 13.4 6 0.8

P P P P P P P P

, 0.0001 ¼ 0.98 ¼ 0.64 ¼ 0.64 ¼ 0.12 ¼ 0.65 ¼ 0.60 , 0.05

Infant Birth weight (g, unadjusted values) Birth weight (g, adjusted values) Placental weight (g) Birth/placental weight ratio Clinically assessed gestational age at birth (wk) Birth length (cm) Head circumference (cm) Abdominal circumference (cm) Sex ratio male/female

3480 6 39 3485 6 20 470 6 10 7.6 6 0.2 38.7 6 0.1 50.5 6 0.1 34.8 6 0.2 34.3 6 0.2 46/44

3167 6 45 3168 6 30 489 6 13 6.7 6 0.2 38.4 6 0.1 48.6 6 0.2 34.5 6 0.2 33.8 6 0.2 32/48

P P P P P P P P P

, 0.0001 , 0.0001 ¼ 0.25 , 0.0001 ¼ 0.10 , 0.0001 ¼ 0.23 , 0.05 ¼ 0.19

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P values

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RESULTS

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FIG. 1. A) PlGF concentrations (pg/ml) were similar at LA (n ¼ 77, median ¼ 223.5, [168.6, 377.0], range 74.0–997.0) versus HA (n ¼ 65, median ¼ 249.4, [152.9, 396.5], range 16.7–862.3, P ¼ 0.87). B) CTAD treatment decreased PlGF by 32% 6 3% (mean 6 SEM) at 400 m (left panel, n ¼ 23) and 33% 6 2% at 3600 m (right panel, n ¼ 48, P , 0.0005 at each altitude). The decrement with CTAD did not differ between altitudes (P ¼ 0.90). C) The serum values for PlGF were positively correlated with placental weight (400 m: y ¼ 92.3 þ 0.82x, r2 ¼ 0.17, P , 0.0005; 3600 m: y ¼68.9 þ 0.76x, r2 ¼ 0.17, P , 0.0001). The slopes (P ¼ 0.64) and intercepts (P ¼ 0.81) did not differ.

not differ between normotensive and preeclamptic pregnancies. The correlation between PlGF and placental weight was significant at both altitudes in PE (Fig. 3C). There was no relationship with gestational age or newborn weight.

the normotensive controls within each altitude. Fetal PO2 was lower in the fetuses of preeclamptic women than controls at both altitudes (400 m, 24 6 2 mmHg, range, 13–35; 3600 m, 23 6 2 mmHg, range, 10–31) (P , 0.01).

Preeclampsia and sFlt-1 Soluble Flt-1 serum levels did not differ between preeclamptic groups at LA versus HA (Fig. 4A), but were greater in PE than normotensive women at both altitudes. Collection into CTAD reduced sFlt-1 levels by 36% 6 5% at 400 m and 42% 6 5% at 3600 m (Fig. 4B). As in the normotensive women, the reduction was quite variable, ranging from 0% to 65% at LA and from 8% to 74% at HA. While these values do not differ from each other, the CTAD-associated reduction in sFlt-1 is greater among preeclamptics than that observed in the normotensive women (41% for all PE vs. 23% for all normotensive, P , 0.0001). There was no difference in sFlt1 levels in early versus late-onset preeclamptic women (Table 2 and Fig. 4, A and B), nor did the CTAD-associated decrement in sFlt-1 vary by severity of disease. Soluble Flt-1 levels were not related to gestational age, birth, or placental weight.

Preeclampsia and PlGF There were no altitude-associated differences in PlGF concentrations among the preeclamptic women, but preeclamptic women had lower PlGF concentrations than their normotensive counterparts (P , 0.001) regardless of altitude (Fig. 3A). Early versus late onset PE did not differ in PlGF concentrations (Table 2 and Fig. 3B). Collection into CTAD reduced the PlGF values in preeclamptics (Fig. 3B; P , 0.001) by 25% 6 5% at LA and by 32% 6 4% at HA (Fig. 3B; P , 0.001). As in the normotensive women, the reduction was quite variable, ranging from 0% to 52% at LA and from 13% to 71% at HA. The decrement was similar between altitudes and did 4

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FIG. 2. A) Soluble Flt-1 concentrations (ng/ml) were similar at LA (n ¼ 76, median ¼ 11.4, [7.0, 20.4], range 2.3–44.4) and HA (n ¼ 74, median ¼ 13.4, [9.0, 21.8], range 1.5–51.4, P ¼ 0.14). B) Collection of blood into CTAD decreased PlGF concentrations by 22% 6 3% at LA and by 23% 6 3% at HA (P , 0.0001 each altitude). The decrement was similar at LA (left panel) versus HA (right panel, P ¼ 0.47).

ANGIOGENIC GROWTH EFFECTORS IN HYPOXIC PREGNANCY TABLE 2. Maternal and infant characteristics in preeclamptic pregnancies. Altitude 400 m Characteristics Maternal Age (yr) Primiparous (n) Height (cm) Nonpregnant weight (kg) Nonpregnant body mass index (kg/m2) Weight gain with pregnancy (kg) Systolic BPb Diastolic BPb Mean arterial pressureb Infant Birth weight (g) Placental weight (g) Birth/placental weight ratio Clinically assessed gestational age at birth (wk) Sex ratio male/female

sFlt-1 (ng/ml) a b

Early-onset PE (n ¼ 7)

Late-onset PE (n ¼ 12)

Early-onset PE (n ¼ 10)

Late-onset PE (n ¼ 10)

26 6 2 6/7 156 6 3 62 6 5 25.4 6 1.8

25 6 1 10/10 160 6 3 62 6 3 24.2 6 1.1

29 6 2 9/10 160 6 2 66 6 5 27.0 6 1.7

27 6 2 9/10 160 6 3 65 6 4 25.4 6 1.6

13.3 6 3.9

16.8 6 1.4

11.6 6 2.5

8.9 6 2.5

P ¼ 0.71

138 6 7 98 6 5 125 6 6

132 6 5 92 6 5 118 6 5

135 6 3 102 6 5 124 6 4

140 6 3 96 6 3 125 6 3

P , 0.0001 P , 0.0001 P , 0.0001

2236 269 8.4 34.3

6 6 6 6

225 35 0.5 0.7

2895 358 8.3 37.0

6 6 6 6

112 27 0.5 0.5

2/5

5/7

145 [121, 179]

172 [148, 272]

98 [29, 174]

47 [11, 121]

1680 316 5.4 33.8

6 6 6 6

161 27 0.4 0.6

6/4

2437 324 8.1 36.7

6 6 6 6

99 35 0.6 0.3

P valuesa P P P P P

P P P P

, 0.005 , 0.0001 ¼ 0.39 ¼ 0.18 ¼ 0.15

, , , ,

0.0001 0.0001 0.0001 0.0001

8/2

157 [87, 274]

144 [93, 220]

57.8 [25.4, 122.3]

24.6 [6.1, 86.0]

P , 0.01 LA; P , 0.001 HA P , 0.0001 LA; P , 0.0001 HA

All the PE are relative to altitude-specific controls. All the women were taking antihypertensive medication, methyldopa, 500 mg/day, which is clinical standard of care in Bolivia. BP, blood pressure.

tensive pregnant women, perhaps accounting for many of the discrepancies in the literature regarding the association (or lack thereof) of sFlt-1 with PE. In serum samples, and possibly in other types of plasma samples, if blood is not processed immediately, this extra PlGF and sFlt-1 appears to be an artifact of blood collection technique. During hemostasis, these growth effectors are likely released by other cell types within the circulation, for example, platelets and granulocytes [29] and other peripheral blood mononuclear cells [30, 31]. Therefore, their alteration in pregnancy pathologies does not necessarily reflect changes in placental production. Challenges to the interpretation of these results include 1) possible differences between the collection sites, 2) the combining of two different ethnic groups at each altitude, 3) assay variability, 4) additional splice variants of sFlt-1, and 5) the lack of gestational age-matched controls for the PE patients. Ambient temperature and barometric pressure were recorded as part of the blood gas measurement protocol. We tested the serum values against ambient room temperature and found no correlation, and nor was any relationship apparent when using cruder measures such as comparing values obtained during summer versus winter. Women of Native American (Andean) ancestry suffer less altitude-associated growth restriction than European migrants. They are not, however, protected from the altitude-associated excess risk of PE [26]. We found no evidence to support ancestry-associated differences in the AGEs, but cannot rule out they may exist between other ethnic groups. In the only other report on Bolivians, altitude decreased sFlt-1 in both native and migrant women and was negatively associated with uterine artery diameter and birth weight in Europeans [36]. We were unable to replicate these findings. Soluble Flt-1 did not differ between ancestry groups or altitudes, and the correlation

Associations Between Variables We tested the hypothesis that variation in maternal and fetal oxygenation would be related to maternal circulating levels of AGEs. We conducted regression analyses of maternal PO2, arterial O2 content, and erythropoietin levels, and of fetal umbilical venous and arterial PO2, O2 content, and erythropoietin concentrations (x axis) with PlGF and sFlt-1 concentrations (y axis). These indicators of maternal and fetal oxygenation showed no relationship to the AGEs, with the r2 values ranging from 0.00 to 0.06. This was true whether it was considered in relation to the entire sample or within each altitude. The hypothesis that variation in circulating angiogenic growth factors might be related to differences in maternal or fetal oxygenation was thus rejected. DISCUSSION The hypotheses tested were not supported. Free PlGF and the soluble binding protein, sFlt-1, did not differ in LA versus HA pregnancies. Surprisingly, given the significant differences in maternal PO2 and SaO2, fetal PO2, and the levels of placental HIF-1alpha and its target genes at HA, multiple indicators of oxygenation were unrelated to circulating levels of AGEs. The extended physiological range of 46 to .100 mmHg arterial PO2 in mothers and of 9–46 mmHg in fetuses was not associated with variation in circulating AGEs. Serum is a widely collected clinical sample. Based on parallel collection of standard serum samples versus immediately processed plasma samples with reagents designed to inhibit platelet activation, we have shown that one-third of PlGF and nearly a quarter of sFlt-1 was secreted/released into blood during hemostasis. Moreover, nearly twice as much sFlt-1 came from peripheral circulatory sources in preeclamptic versus normo5

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Serum AGF concentrations (median, [interquartile range]) PlGF (pg/ml)

Altitude 3600 m

ZAMUDIO ET AL.

contribute to heterogeneity in the results reported in the relevant literature. Much of what we know about the interaction between the AGEs, vascular endothelium, and disease states is not consistent with the pattern argued as a causal contributor to PE. In theory, lower free VEGF should be beneficial because high levels of free VEGF in nonpregnant persons are associated with hypertension, glomerulosis, altered vascular reactivity, and vascular leak, all the hallmarks of PE [39–43]. Excess PlGF, like VEGF, is implicated in hypertension and vascular disease [44]. Stretch, shear stress, hypoxia, and proinflammatory stimuli will induce PlGF expression [45–49]. PlGF is known to stimulate monocyte production of proinflammatory cytokines [50] that are elevated in PE [51]. Hence, the evidence favors increased, not decreased, PlGF as a causal component of PE. Placental size was correlated with PlGF in both our normal and preeclamptic pregnancies. However the absence of excess PlGF in twin pregnancy argues against placental mass as a primary determinant. The rise in PlGF in the maternal

coefficient (r2) for uterine artery diameter and sFlt-1 was 0.05 and for birth weight was 0.08 to altitude among migrants. These differences, and the high variability of reported values for AGEs in the literature cast doubt on the utility of these measures for clinical diagnoses. We are aware that additional splice variants of sFlt-1 have been discovered [37] and are produced by the hypoxic placental trophoblast [7, 38] and that it is unknown whether current assays measuring sFlt-1 can distinguish between these splice variants. Finally, the lack of age-matched controls for the PE patients is a weakness, but it was not possible to collect such samples in the absence of some other pathology. In summary, variability in, among other factors, the assays themselves, the testing conditions, blood collection protocols, and length of storage, if not accounted for, are all likely to 6

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FIG. 4. A) Open squares and diamond symbols are early and late onset PE, respectively, at LA; filled squares and diamonds are early and late onset PE, respectively at HA. Soluble Flt-1 serum levels (ng/ml) were greater than in normotensive women at each altitude but did not differ in preeclamptic women at LA (median ¼ 43) [16, 97] versus HA (median ¼ 49; P ¼ 0.49) [27, 133]. B) Collection into CTAD reduced sFlt-1 levels by 36% 6 5% at 400 m and 42% 6 5% at 3600 m (P , 0.005). While these values do not differ from each other, the CTAD-associated reduction in sFlt-1 is significantly greater among preeclamptics than what was observed in the normotensive women (P , 0.0001).

FIG. 3. A) Open squares and diamond symbols are early and late onset PE, respectively, at LA; filled squares and diamonds are early and late onset PE, respectively, at HA. PlGF values (pg/ml) were lower in preeclamptic than normotensive women at both LA (median ¼ 168) [133, 236] and HA (median ¼ 144) [92, 240], but there was no altitudeassociated difference in the values for PlGF in preeclamptics. B) Collection of samples into CTAD reduced the values for PlGF in preeclamptic women at both 400 m (right panel) and 3600 m (left panel). C) As with the normotensive control pregnancies, PlGF concentrations in preeclamptics correlated with placental weight (400 m: y ¼ 0.533x þ 3.953, r2 ¼ 0.47, P , 0.005; 3600 m: y ¼ 0.557x  17.64, r2 ¼ 0.40, P , 0.005). The slopes do not differ (P ¼ 0.32).

ANGIOGENIC GROWTH EFFECTORS IN HYPOXIC PREGNANCY

ACKNOWLEDGMENT The extraordinary generosity and support of the following physiciancolleagues in Bolivia made this work possible: Dra. Maria Luz Almendros Pasantino, Dr. Julio Ameller, Dr. Tatiana Alvarez, Dr. Gonzalo Azurdy, Dra. Elfrida Balanza, Dra. Diva Bellido, Mr. Christian Cerusoli, Dra. Edith Claros Mercado, Dra. Yohelma Eid, Dr. Carlos Loayza, Mr. Ivan Maldonado, Dr. Fernando Meswith, Dra. Lucretia Postigo, Dra. Jesica Pardo Quiroga, Dra. Lilian Toledo, and Dr. Marco Vargas.

REFERENCES 1. Lyall F, Young A, Boswell F, Kingdom JC, Greer IA. Placental expression of vascular endothelial growth factor in placentae from pregnancies complicated by pre-eclampsia and intrauterine growth restriction does not support placental hypoxia at delivery. Placenta 1997; 18:269–276. 2. Vuorela P, Helske S, Hornig C, Alitalo K, Weich H, Halmesmaki E. Amniotic fluid—soluble vascular endothelial growth factor receptor-1 in preeclampsia. Obstet Gynecol 2000; 95:353–357. 3. Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, Libermann TA, Morgan JP, Sellke FW, Stillman IE, Epstein FH, Sukhatme VP, et al. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest 2003; 111:649–658. 4. Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF, Schisterman EF, Thadhani R, Sachs BP, Epstein FH, Sibai BM, Sukhatme VP, et al. Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med 2004; 350:672–683. 5. Caniggia I, Taylor CV, Ritchie JW, Lye SJ, Letarte M. Endoglin regulates trophoblast differentiation along the invasive pathway in human placental villous explants. Endocrinology 1997; 138:4977–4988. 6. Venkatesha S, Toporsian M, Lam C, Hanai J, Mammoto T, Kim YM,

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found changes prior to symptom onset only in individuals at known high risk [62, 63]. Negative publication bias may have limited the numbers of reports in which no predictive value was shown. Others have shown that the values obtained have low sensitivity and specificity [64, 65], and are unrelated to markers of endothelial cell dysfunction [66], and that changes are due to an increase in blood pressure rather than the reverse [54]. The significant overlap between the values obtained in normotensive versus preeclamptic pregnancy reported here and elsewhere, combined with the variable change in the degree to which activation of peripheral cells contributes to the values measured, suggests that circulating values are unlikely to attain a sufficient degree of specificity and sensitivity for use as a reliable diagnostic test. Finally, our results argue against hypoxia as specifically, causally associated with changes in circulating angiogenic growth factors: lowered maternal or fetal PO2 does not appear to cause change in tissue production and release into the circulation of the angiogenic growth factors tested here. In summary, we have shown that multiple measures of maternal and fetal hypoxia are not related to circulating AGEs in human pregnancy. The hypothesis that placental hypoxia increases secretion of sFlt-1 into the maternal circulation is not supported by these data. Standard serum samples do not accurately reflect the actual levels of circulating AGEs in pregnant women. That the error is much greater for serum levels of sFlt-1 in pregnancies complicated by PE points to one possible explanation for discrepancies in the literature regarding the extent to which elevated sFlt-1 may or may not precede development of symptoms. This casts doubt on the theory that excess circulating sFlt-1 is causally associated with the development of symptoms. More fruitful research might focus on how cells in the peripheral circulation interact with the maternal endothelium and to what extent highly localized release of growth factors may influence endothelial cell function to protect against or exacerbate preeclamptic symptoms.

circulation in the early second trimester is exponential, correlates with Doppler indicators of placental perfusion, and likely reflects placental perfusion as well as increase in mass [52]. Alternatively, opening of the maternal intervillous space to blood flow at ;10–12 wk may induce PlGF due to shear stress or stretch [46, 47], which is largely complete by 32 wk, thereafter removing the stretch stimulus, which may be why PlGF declines in the third trimester. In either case, hypoperfusion due to impaired development of the spiral arteries and/or failed growth of the placenta, common in severe PE, may account for the relatively lower levels of PlGF in preeclamptic pregnancy without necessarily having any impact on the maternal endothelium or on the development of symptoms. Again the question must be asked, what does a relative paucity of PlGF actually do to endothelial cell health in pregnancy? Soluble Flt-1 is a splice variant of VEGF receptor 1. Production by the placenta was first reported in 1998 [53]; reports on the elevation of sFlt-1 in PE and by hypoxia followed in 2000 [2, 18]. In normal pregnancy, but not PE, the rise in sFlt-1 correlates with the third trimester rise in blood pressure [54]. We found no association with mean arterial pressure in this study (r2 values ranged from 0.00 to 0.04 within and between altitudes). Nonetheless, antihypertensive therapy reduces sFlt-1 in preeclamptics [55]. As with VEGF and PlGF, the damaging role claimed for elevated sFlt-1 in preeclamptic pregnancy is counterintuitive. Soluble Flt-1 is lower in hypertensive men and elevated by therapies designed to ameliorate cardiovascular risk. It is worth noting that PlGF values in PE overlap entirely with those of normotensive women, while sFlt-1 levels overlap in at least half the cases (Figs. 3 and 4). The source of the excess sFlt-1 in PE has not been fully established. We show here, in theory, that excess placental production is not reflected in the maternal circulation with physiological hypoxia. Alternatively, if there is excess placental production in PE, not due to hypoxia, it may be bound by peripheral cells in the circulation and contribute to the higher values of sFlt-1 in preeclamptic women. Preeclampsia is associated with failure of plasma volume expansion, which may contribute to higher sFlt-1 levels resulting simply from hemoconcentration. However, if this was the case, we would expect to see elevated sFlt-1 at HA because plasma volume is lower in both the nonpregnant and pregnant state [56]. Cells other than platelets must be involved because HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome, characterized by thrombocytopenia, has AGE profiles similar to PE whereas idiopathic thrombocytopenia in pregnancy is associated with lower sFlt-1 and higher PlGF than normal pregnancy [57]. Altered levels of peripheral blood mononuclear cells, activation of platelets (which are normally reduced), and altered leucocyte populations in the maternal decidua have all been reported in PE [58] and may also contribute to variation in circulating concentrations of AGEs. Our results support the accumulating evidence in the literature that the AGEs studied here play a correlative rather than causal role in PE. It is at least possible that elevated sFlt-1 is a protective response to excess free VEGF production by the hypoxic placenta. It is noteworthy that free VEGF decreases by an order of magnitude or more from the nonpregnant state and that, unlike in rodents, which are common models for PE, there is basically no free VEGF circulating in human pregnancy. A recent National Institutes of Health trial revealed that first trimester values of AGEs have little predictive value [59] even if supplemented with Doppler measures of resistance. Some groups have shown changes in the mean values of maternal circulating angiogenic growth factors several weeks prior to the onset of symptoms [4, 60, 61], while others have not, or have

ZAMUDIO ET AL.

7.

8.

9.

10.

11. 12.

13.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

8

Article 42

Downloaded from www.biolreprod.org.

14.

27. Zamudio S. High-altitude hypoxia and preeclampsia. Front Biosci 2007; 12:2967–2977. 28. Zamudio S, Kovalenko O, Echalar L, Torricos T, Al-Khan A, Alvarez M, Illsley NP. Evidence for extraplacental sources of circulating angiogenic growth effectors in human pregnancy. Placenta 2013; 34:1170–1176. 29. Vuorela P, Lintula S, Stenman UH, Halmesmaki E. Expression of vascular endothelial growth factor in peripheral blood cells of preeclamptic women. Hypertens Pregnancy 2003; 22:193–201. 30. Rajakumar A, Michael HM, Rajakumar PA, Shibata E, Hubel CA, Karumanchi SA, Thadhani R, Wolf M, Harger G, Markovic N. Extraplacental expression of vascular endothelial growth factor receptor-1, (Flt1) and soluble Flt-1 (sFlt-1), by peripheral blood mononuclear cells (PBMCs) in normotensive and preeclamptic pregnant women. Placenta 2005; 26:563–573. 31. Makris A, Thornton C, Thompson J, Thomson S, Martin R, Ogle R, Waugh R, McKenzie P, Kirwan P, Hennessy A. Uteroplacental ischemia results in proteinuric hypertension and elevated sFLT-1. Kidney Int 2007; 71:977–984. 32. Roberts JM, Pearson G, Cutler J, Lindheimer M. Summary of the NHLBI Working Group on Research on Hypertension During Pregnancy. Hypertension 2003; 41:437–445. 33. Webb NJ, Bottomley MJ, Watson CJ, Brenchley PE. Vascular endothelial growth factor (VEGF) is released from platelets during blood clotting: implications for measurement of circulating VEGF levels in clinical disease. Clin Sci (Lond) 1998; 94:395–404. 34. Jelkmann W. Pitfalls in the measurement of circulating vascular endothelial growth factor. Clin Chem 2001; 47:617–623. 35. Dittadi R, Meo S, Fabris F, Gasparini G, Contri D, Medici M, Gion M. Validation of blood collection procedures for the determination of circulating vascular endothelial growth factor (VEGF) in different blood compartments. Int J Biol Markers 2001; 16:87–96. 36. Davila RD, Julian CG, Wilson MJ, Browne VA, Rodriguez C, Bigham AW, Shriver MD, Vargas E, Moore LG. Do anti-angiogenic or angiogenic factors contribute to the protection of birth weight at high altitude afforded by Andean ancestry? Reprod Sci 2010; 17:861–870. 37. Sela S, Itin A, Natanson-Yaron S, Greenfield C, Goldman-Wohl D, Yagel S, Keshet E. A novel human-specific soluble vascular endothelial growth factor receptor 1: cell-type-specific splicing and implications to vascular endothelial growth factor homeostasis and preeclampsia. Circ Res 2008; 102:1566–1574. 38. Thomas CP, Andrews JI, Raikwar NS, Kelley EA, Herse F, Dechend R, Golos TG, Liu KZ. A recently evolved novel trophoblast-enriched sFlt1 variant is upregulated in hypoxia and in preeclampsia. J Clin Endocrinol Metab 2009; 94:2524–2530. 39. Shoab SS, Scurr JH, Coleridge-Smith PD. Increased plasma vascular endothelial growth factor among patients with chronic venous disease. J Vasc Surg 1998; 28:535–540. 40. Kakizawa H, Itoh M, Itoh Y, Imamura S, Ishiwata Y, Matsumoto T, Yamamoto K, Kato T, Ono Y, Nagata M, Hayakawa N, Suzuki A, et al. The relationship between glycemic control and plasma vascular endothelial growth factor and endothelin-1 concentration in diabetic patients. Metabolism 2004; 53:550–555. 41. Belgore FM, Blann AD, Li-Saw-Hee FL, Beevers DG, Lip GY. Plasma levels of vascular endothelial growth factor and its soluble receptor (SFlt1) in essential hypertension. Am J Cardiol 2001; 87:805–807. 42. Felmeden DC, Spencer CG, Belgore FM, Blann AD, Beevers DG, Lip GY. Endothelial damage and angiogenesis in hypertensive patients: relationship to cardiovascular risk factors and risk factor management. Am J Hypertens 2003; 16:11–20. 43. Nadar SK, Blann AD, Lip GY. Plasma and platelet-derived vascular endothelial growth factor and angiopoietin-1 in hypertension: effects of antihypertensive therapy. J Intern Med 2004; 256:331–337. 44. Briguori C, Testa U, Colombo A, Petrucci E, Condorelli G, Airoldi F, Peschle C, Condorelli G. Relation of various plasma growth factor levels in patients with stable angina pectoris and total occlusion of a coronary artery to the degree of coronary collaterals. Am J Cardiol 2006; 97: 472–476. 45. Cramer M, Nagy I, Murphy BJ, Gassmann M, Hottiger MO, Georgiev O, Schaffner W. NF-kappaB contributes to transcription of placenta growth factor and interacts with metal responsive transcription factor-1 in hypoxic human cells. Biol Chem 2005; 386:865–872. 46. Werner GS, Jandt E, Krack A, Schwarz G, Mutschke O, Kuethe F, Ferrari M, Figulla HR. Growth factors in the collateral circulation of chronic total coronary occlusions: relation to duration of occlusion and collateral function. Circulation 2004; 110:1940–1945. 47. Mohammed KA, Nasreen N, Tepper RS, Antony VB. Cyclic stretch induces PlGF expression in bronchial airway epithelial cells via nitric

Bdolah Y, Lim KH, Yuan HT, Libermann TA, Stillman IE, Roberts D, et al. Soluble endoglin contributes to the pathogenesis of preeclampsia. Nat Med 2006; 12:642–649. Rajakumar A, Powers RW, Hubel CA, Shibata E, von Versen-Hoynck F, Plymire D, Jeyabalan A. Novel soluble Flt-1 isoforms in plasma and cultured placental explants from normotensive pregnant and preeclamptic women. Placenta 2009; 30:25–34. Whitehead CL, Palmer KR, Nilsson U, Gao Y, Saglam B, Lappas M, Tong S. Placental expression of a novel primate-specific splice variant of sFlt-1 is upregulated in pregnancies complicated by severe early onset preeclampsia. BJOG 2011; 118:1268–1271. Goldman CK, Kendall RL, Cabrera G, Soroceanu L, Heike Y, Gillespie GY, Siegal GP, Mao X, Bett AJ, Huckle WR, Thomas KA, Curiel DT. Paracrine expression of a native soluble vascular endothelial growth factor receptor inhibits tumor growth, metastasis, and mortality rate. Proc Natl Acad Sci U S A 1998; 95:8795–8800. Roberts JM, Taylor RN, Musci TJ, Rodgers GM, Hubel CA, McLaughlin MK. Preeclampsia: an endothelial cell disorder [see comments]. Am J Obstet Gynecol 1989; 161:1200–1204. Wheeler T, Elcock CL, Anthony FW. Angiogenesis and the placental environment. Placenta 1995; 16:289–296. Taylor CM, Stevens H, Anthony FW, Wheeler T. Influence of hypoxia on vascular endothelial growth factor and chorionic gonadotrophin production in the trophoblast-derived cell lines: JEG, JAr and BeWo. Placenta 1997; 18:451–458. Hornig C, Barleon B, Ahmad S, Vuorela P, Ahmed A, Weich HA. Release and complex formation of soluble VEGFR-1 from endothelial cells and biological fluids. Lab Invest 2000; 80:443–454. Khaliq A, Dunk C, Jiang J, Shams M, Li XF, Acevedo C, Weich H, Whittle M, Ahmed A. Hypoxia down-regulates placenta growth factor, whereas fetal growth restriction up-regulates placenta growth factor expression: Molecular evidence for ‘‘placental hyperoxia’’ in intrauterine growth restriction. Lab Invest 1999; 79:151–170. Li H, Gu B, Zhang Y, Lewis DF, Wang Y. Hypoxia-induced increase in soluble Flt-1 production correlates with enhanced oxidative stress in trophoblast cells from the human placenta. Placenta 2005; 26:210–217. Soleymanlou N, Jurisica I, Nevo O, Ietta F, Zhang X, Zamudio S, Post M, Caniggia I. Molecular evidence of placental hypoxia in preeclampsia. J Clin Endocrinol Metab 2005; 90:4299–4308. Muttukrishna S, Swer M, Suri S, Jamil A, Calleja-Agius J, Gangooly S, Ludlow H, Jurkovic D, Jauniaux E. Soluble Flt-1 and PlGF: new markers of early pregnancy loss? PLoS One 2011; 6:e18041. Ahmed A, Dunk C, Ahmad S, Khaliq A. Regulation of placental vascular endothelial growth factor (VEGF) and placenta growth factor (PIGF) and soluble Flt-1 by oxygen—a review. Placenta 2000; 21(Suppl A):S16–S24. Munaut C, Lorquet S, Pequeux C, Blacher S, Berndt S, Frankenne F, Foidart JM. Hypoxia is responsible for soluble vascular endothelial growth factor receptor-1 (VEGFR-1) but not for soluble endoglin induction in villous trophoblast. Hum Reprod 2008; 23:1407–1415. Nevo O, Soleymanlou N, Wu Y, Xu J, Kingdom J, Many A, Zamudio S, Caniggia I. Increased expression of sFlt-1 in in vivo and in vitro models of human placental hypoxia is mediated by HIF-1. Am J Physiol Regul Integr Comp Physiol 2006; 291:R1085–R1093. Rajakumar A, Brandon HM, Daftary A, Ness R, Conrad KP. Evidence for the functional activity of hypoxia-inducible transcription factors overexpressed in preeclamptic placentae. Placenta 2004; 25:763–769. Zamudio S, Wu Y, Ietta F, Rolfo A, Cross A, Wheeler T, Post M, Illsley NP, Caniggia I. Human placental hypoxia-inducible factor-1alpha expression correlates with clinical outcomes in chronic hypoxia in vivo. Am J Pathol 2007; 170:2171–2179. Zamudio S, Postigo L, Illsley NP, Rodriguez C, Heredia G, Brimacombe M, Echalar L, Torricos T, Tellez W, Maldonado I, Balanza E, Alvarez T, et al. Maternal oxygen delivery is not related to altitude- and ancestryassociated differences in human fetal growth. J Physiol 2007; 582: 883–895. Postigo L, Heredia G, Illsley NP, Torricos T, Dolan C, Echalar L, Tellez W, Maldonado I, Brimacombe M, Balanza E, Vargas E, Zamudio S. Where the O2 goes to: preservation of human fetal oxygen delivery and consumption at high altitude. J Physiol 2009; 587:693–708. Palmer SK, Moore LG, Young D, Cregger B, Berman JC, Zamudio S. Altered blood pressure course during normal pregnancy and increased preeclampsia at high altitude (3100 meters) in Colorado. Am J Obstet Gynecol 1999; 180:1161–1168. Keyes LE, Armaza JF, Niermeyer S, Vargas E, Young DA, Moore LG. Intrauterine growth restriction, preeclampsia, and intrauterine mortality at high altitude in Bolivia. Pediatr Res 2003; 54:20–25.

ANGIOGENIC GROWTH EFFECTORS IN HYPOXIC PREGNANCY

48. 49.

50. 51. 52.

53.

54.

55.

57.

58.

59. Myatt L, Clifton RG, Roberts JM, Spong CY, Hauth JC, Varner MW, Thorp JM Jr, Mercer BM, Peaceman AM, Ramin SM, Carpenter MW, Iams JD, et al. First-trimester prediction of preeclampsia in nulliparous women at low risk. Obstet Gynecol 2012; 119:1234–1242. 60. Levine RJ, Thadhani R, Qian C, Lam C, Lim KH, Yu KF, Blink AL, Sachs BP, Epstein FH, Sibai BM, Sukhatme VP, Karumanchi SA. Urinary placental growth factor and risk of preeclampsia. JAMA 2005; 293:77–85. 61. Romero R, Nien JK, Espinoza J, Todem D, Fu W, Chung H, Kusanovic JP, Gotsch F, Erez O, Mazaki-Tovi S, Gomez R, Edwin S, et al. A longitudinal study of angiogenic (placental growth factor) and antiangiogenic (soluble endoglin and soluble vascular endothelial growth factor receptor-1) factors in normal pregnancy and patients destined to develop preeclampsia and deliver a small for gestational age neonate. J Matern Fetal Neonatal Med 2008; 21:9–23. 62. Savvidou MD, Yu CK, Harland LC, Hingorani AD, Nicolaides KH. Maternal serum concentration of soluble fms-like tyrosine kinase 1 and vascular endothelial growth factor in women with abnormal uterine artery Doppler and in those with fetal growth restriction. Am J Obstet Gynecol 2006; 195:1668–1673. 63. Powers RW, Jeyabalan A, Clifton RG, Van Dorsten P, Hauth JC, Klebanoff MA, Lindheimer MD, Sibai B, Landon M, Miodovnik M, Eunice Kennedy Shriver National Institute of Child Health Human Development Maternal-Fetal Medicine Units Network. Soluble fms-Like tyrosine kinase 1 (sFlt1), endoglin and placental growth factor (PlGF) in preeclampsia among high risk pregnancies. PLoS One 2010; 5:e13263. 64. Sibai BM, Koch MA, Freire S, Pinto e Silva JL, Rudge MV, MartinsCosta S, Bartz J, de Barros Santos C, Cecatti JG, Costa R, Ramos JG, Spinnato JA II. Serum inhibin A and angiogenic factor levels in pregnancies with previous preeclampsia and/or chronic hypertension: are they useful markers for prediction of subsequent preeclampsia? Am J Obstet Gynecol 2008; 199(268):e261–e269. 65. Krauss T, Pauer HU, Augustin HG. Prospective analysis of placenta growth factor (PlGF) concentrations in the plasma of women with normal pregnancy and pregnancies complicated by preeclampsia. Hypertens Pregnancy 2004; 23:101–111. 66. Savvidou MD, Noori M, Anderson JM, Hingorani AD, Nicolaides KH. Maternal endothelial function and serum concentrations of placental growth factor and soluble endoglin in women with abnormal placentation. Ultrasound Obstet Gynecol 2008; 32:871–876.

9

Article 42

Downloaded from www.biolreprod.org.

56.

oxide release. Am J Physiol Lung Cell Mol Physiol 2007; 292: L559–L566. Chang M, Mukherjea D, Gobble RM, Groesch KA, Torry RJ, Torry DS. Glial cell missing 1 regulates placental growth factor (PGF) gene transcription in human trophoblast. Biol Reprod 2008; 78:841–851. Bahtiyar MO, Buhimschi C, Ravishankar V, Copel J, Norwitz E, Julien S, Guller S, Buhimschi IA. Contrasting effects of chronic hypoxia and nitric oxide synthase inhibition on circulating angiogenic factors in a rat model of growth restriction. Am J Obstet Gynecol 2007; 196(72):e71–e76. Selvaraj SK, Giri RK, Perelman N, Johnson C, Malik P, Kalra VK. Mechanism of monocyte activation and expression of proinflammatory cytochemokines by placenta growth factor. Blood 2003; 102:1515–1524. Keelan JA, Mitchell MD. Placental cytokines and preeclampsia. Front Biosci 2007; 12:2706–2727. Welch PC, Amankwah KS, Miller P, McAsey ME, Torry DS. Correlations of placental perfusion and PlGF protein expression in early human pregnancy. Am J Obstet Gynecol 2006; 194:1625–1629. Discussion 1629–1631. Clark DE, Smith SK, He Y, Day KA, Licence DR, Corps AN, Lammoglia R, Charnock-Jones DS. A vascular endothelial growth factor antagonist is produced by the human placenta and released into the maternal circulation. Biol Reprod 1998; 59:1540–1548. Troisi R, Braekke K, Harsem NK, Hyer M, Hoover RN, Staff AC. Blood pressure augmentation and maternal circulating concentrations of angiogenic factors at delivery in preeclamptic and uncomplicated pregnancies. Am J Obstet Gynecol 2008; 199(653):e651–e610. Khalil A, Muttukrishna S, Harrington K, Jauniaux E. Effect of antihypertensive therapy with alpha methyldopa on levels of angiogenic factors in pregnancies with hypertensive disorders. PLoS ONE 2008; 3: e2766. Zamudio S, Palmer SK, Dahms TE, Berman JC, McCullough RG, McCullough RE, Moore LG. Blood volume expansion, preeclampsia, and infant birth weight at high altitude. J Appl Physiol 1993; 75:1566–1573. Young B, Levine RJ, Salahuddin S, Qian C, Lim KH, Karumanchi SA, Rana S. The use of angiogenic biomarkers to differentiate non-HELLP related thrombocytopenia from HELLP syndrome. J Matern Fetal Neonatal Med 2010; 23:366–370. Williams PJ, Bulmer JN, Searle RF, Innes BA, Robson SC. Altered decidual leucocyte populations in the placental bed in pre-eclampsia and foetal growth restriction: a comparison with late normal pregnancy. Reproduction 2009; 138:177–184.