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Am J Physiol Regul Integr Comp Physiol 293: R766-R774, 2007. First published May 16, 2007; doi:10.1152/ajpregu.00097.2007
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DEVELOPMENTAL PHYSIOLOGY AND PREGNANCY

Placental HIF-1{alpha}, HIF-2{alpha}, membrane and soluble VEGF receptor-1 proteins are not increased in normotensive pregnancies complicated by late-onset intrauterine growth restriction

Augustine Rajakumar,1 Arun Jeyabalan,1 Nina Markovic,2 Roberta Ness,2 Carol Gilmour,3 and Kirk P. Conrad1,4

Departments of 1Obstetrics, Gynecology and Reproductive Sciences, and 2Epidemiology, Graduate School of Public Health and 4Cell Biology and Physiology, University of Pittsburgh School of Medicine and Magee Womens Research Institute Pittsburgh and 3Pediatrics, Allegheny General Hospital and Drexel University School of Medicine, Pittsburgh, Pennsylvania

Submitted 12 February 2007 ; accepted in final form 14 May 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Inadequate trophoblast invasion and spiral artery remodeling leading to poor placental perfusion are believed to underlie the pregnancy pathologies preeclampsia (PE) and intrauterine growth restriction (IUGR). The main objective of this study was to investigate hypoxia-inducible transcription factor-{alpha} (HIF-{alpha}) and downstream genes (VEGF receptor-1) Flt-1 and soluble fms-like tyrosine kinase 1 (sFlt-1) proteins in IUGR placentas. Placentas from normal pregnant (NP; n = 18), PE (n = 18), and IUGR (n = 10) patients were investigated. Normotensive patients with IUGR delivered babies at ≥ 37 wk of gestation with birth weights of <10% and asymmetrical growth. HIF-1{alpha}, -2{alpha}, Flt-1, and sFlt-1 protein, and mRNA were assessed by Western and Northern blot analyses, respectively. The results are expressed as ratios of the densitometric values for each pair of pathologic and normal placentas, a ratio of 1.0 indicating no difference. Comparable to our earlier studies, the PE/NP ratios for HIF-1{alpha}, -2{alpha}, and Flt proteins were significantly increased by 50–100% (all P < 0.01 vs. 1.0). Unexpectedly, the IUGR/NP ratios for HIF-1{alpha} and -2{alpha} proteins were 1.03 ± 0.07 and 0.96 ± 0.16, respectively, and for Flt and sFlt were 1.14 ± 0.15 and 0.95 ± 0.12, respectively (all P = not significant vs. 1.0). Northern blot analysis revealed comparable levels of HIF-{alpha} mRNA in abnormal and normal placentas. In contrast to PE, HIF-{alpha} proteins and regulated genes are not increased in placentas from normotensive pregnant women delivering small, asymmetrically grown babies ≥ 37 wk of gestation. The absence of an increase in HIF-{alpha} protein is not due to insufficient HIF-{alpha} mRNA for protein synthesis. Thus, the placentas from women with PE and late IUGR are fundamentally different at the molecular level.

preeclampsia; fetal growth restriction; placenta; hypoxia-inducible transcription factors; Flt-1

INADEQUATE TROPHOBLAST INVASION and remodeling of uterine spiral arteries leading to poor placental perfusion is believed to underlie the pregnancy pathologies preeclampsia and intrauterine growth restriction (IUGR) (18, 21). We previously documented that hypoxia-inducible transcription factors-1{alpha} (HIF-1{alpha}) and -2{alpha} proteins, and target genes, such as Flt-1 and tyrosine hydroxylase are significantly increased in preeclampsia placentas by 50–100% relative to normal-term and gestationally aged-matched control placentas (27, 29). This placental overexpression of HIF-{alpha} protein and downstream genes is most likely due to several factors, including local hypoxia (38) as well as a defect in oxygen-dependent degradation of HIF-{alpha} protein (28). In the present study, we investigated placentas obtained from normotensive women who delivered growth-restricted babies (idiopathic IUGR). Since preeclampsia pregnancies (with or without IUGR) and idiopathic IUGR are believed to share the same placental etiology, i.e., inadequate trophoblast invasion and spiral artery remodeling leading to poor placental perfusion, and consequently, decreased oxygen delivery, we hypothesized that the expression of HIF-{alpha} and target genes Flt-1 and soluble fms-like tyrosine kinase 1 (sFlt-1) (38), would be increased in placentas from normotensive women delivering growth-restricted fetuses comparable to that observed in placentas from women with preeclampsia (19, 27, 29). In this work, we focused on late-onset IUGR because being more frequent than early-onset IUGR representing 90 and 10% of IUGR cases (5), respectively, our sample collection reflected this distribution and consisted almost exclusively of late-onset IUGR cases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Placental collection and processing. After informed consent and under the approval of the Institutional Internal Review Board of the University of Pittsburgh, placentas were obtained from women with normal (n = 18) and preeclamptic (n = 18) pregnancies, as well as from women who delivered small babies (n = 14). The placentas were sampled immediately after extraction from the uterus. To ensure systematic and unbiased sampling of the entire placenta, we fitted a circular plastic grid containing 16 holes over the maternal face of the placenta and obtained 16 biopsy samples (4, 29). Each ~0.5-g sample contained the decidua basalis and villous placenta but not the chorionic plate. After several rapid rinses in chilled saline, the tissues were blotted dry and flash frozen in liquid nitrogen. In this study, we randomly chose three biopsy sites from the 16-site biopsy collection, and pooled the three biopsy sites, each of comparable mass, to represent a single placenta.

Clinical definitions. Clinical details for the patient groups are shown in Table 1. The diagnosis of preeclampsia was made based on the Working Group Report on High Blood Pressure in Pregnancy (30). Gestational blood pressure elevation was defined as systolic blood pressure of ≥140 or diastolic pressure of ≥90 mmHg. Furthermore, the subjects were normotensive during early pregnancy or postpartum with no history of chronic hypertension. Preeclamptic subjects had new-onset proteinuria of >2+ on dipstick or urinary creatinine/protein ratio of >0.3, and hyperuricemia of 1 SD above normal for gestational age.


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  		Table 1. General clinical parameters of all subjects

 
As shown in Table 2, five of the 18 preeclamptic subjects delivered small babies (≤5.5% based on gestational age at delivery, sex, and race for Magee-Womens Hospital), all of whom were asymmetrically grown (see below) and/or showed abnormal umbilical artery Doppler waveforms. The umbilical artery Doppler was considered to be abnormal if the systolic-to-diastolic ratio was greater than the 95% for gestational age or if the diastolic flow was absent or reversed (9a). Thus, we considered these babies to be pathologically small or growth restricted rather than constitutionally small babies.


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  		Table 2. Clinical characteristics of preeclamptic pregnancies with small babies

 
Fourteen patients without preeclampsia or gestational hypertension delivered small babies with weights < 10% based on gestational age at delivery, sex, and race for Magee-Womens Hospital. Patients with underlying medical disorders, infection during pregnancy, or substance abuse, including smoking, were excluded. There were no recognized chromosomal or genetic defects in the babies. Symmetrically small babies showed proportionate weight, length, and head circumference below the population 10th percentile. Asymmetric IUGR was defined by birth weight < length ≤ head circumference percentiles when the weight percentile was at least two percentile categories below length and/or head circumference. Percentile categories were defined by cut points at 3, 5, 10, 25, 50, 75, 90, 95, 97% (32). For example, the infant born of patient 6 in Table 3 has a birth percentile of 2.0 (category < 3), length percentile of 10–25, and head circumference percentile of 10. Thus, the weight percentile is at least two categories below length and below the head circumference percentiles. Ten of 14 manifested asymmetric growth, and thus we considered these to be pathologically small or growth-restricted babies (Table 3). In addition, seven of these 14 babies were ≤ 2.4%, a baby weight more likely to be associated with absent trophoblast invasion and physiological remodeling of spiral arteries in the placental bed (13).


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  		Table 3. Clinical characteristics of normotensive pregnancies with small babies

 
Placental weights were obtained for most of the patients and are listed in the tables. The placenta percentile weight was determined based on Magee-Womens Hospital data for gestational ages 18–43 wk. The percentile ranges are <10, 10–25, 25–50, 50–75, 75–90, and >90.

Western blot analysis. Total protein was extracted and Western blot analysis was conducted using our published procedures with minor modification (27, 29). An anti-HIF-1{alpha} monoclonal antibody (cat. no. H72320 [GenBank] ; Transduction Laboratories, Lexington, KY) and a rabbit polyclonal anti-HIF-2{alpha} antibody (cat. no. NB 100–122B2; Novus Biologicals, Littleton, CO) were utilized. The HIF-1{alpha} antibody was diluted 1:200 in Tris-buffered saline containing 0.05% Tween-20 (1.25 µg/ml); the HIF-2{alpha} antibody was diluted 1:1,000 (2.2 µg/ml). Rabbit anti-human Flt-1 (cat. no. SC-316; Santa Cruz Biotechnology, Santa Cruz, CA) was used to detect the full-length 185-kDa Flt-1 and mouse monoclonal anti-human Flt-1 (cat. no. V4262; Sigma, St Louis, MO) was used to detect the 100-kDa sFlt-1. They were diluted 1:200 and 1:1,000, respectively, each to 2.2 µg/ml. The mouse monoclonal anti-beta-actin antibody (cat. no. A 5441; Sigma) was diluted 1:1,000 yielding final concentrations of 2.2 µg/ml. Fifty micrograms total protein was electrophoresed for detection of HIF-{alpha}, Flt-1, and sFlt-1. For the analysis of beta-actin, 5 µg of total protein was electrophoresed on separate gels and subjected to Western blot analysis. In addition, Coomassie blue staining of gels and membranes indicated uniform loading and transfer (data not shown).

Northern blot analysis. Total RNA was isolated from placental tissues (n = 8 for each group) using RNAwiz (Ambion, Austin, TX) and Northern blot analysis was conducted using our previously published procedures (29). For the preparation of the cDNA probes, plasmids pBS/HIF-1{alpha} 3.2–3T7 (EcoRI digest of the complete cDNA produces three bands of sizes 2,063, 1,011, and 604 bp) was generously provided by Dr. Gregg Semenza. Plasmid hEPAS-pcDNA3 (HIF-2{alpha}, a 2,818-bp BamHI fragment) was a kind gift from Dr. Steven McKnight. A 376-bp DNA fragment specific for human beta-actin (GenBank accession no. X00351) was amplified using RT-PCR (forward primer 5'-AGCCATGTACGTTGCTATCCAGGCTGTGCT-3' and reverse primer 5'-AGCGGAACCGCTCATTGCCAATGGTGATGA-3').

Densitometry. The bands on the developed Western and Northern blot films, as well as the bands on the ethidium bromide-stained gel (for 18S RNA), were scanned using a laser scanner (Scanjet 5370C, Hewlett Packard, Palo Alta, CA) into a PICT or TIFF file in grey scale. Densitometry was performed using an automated digitizing software UN-SCANIT gel (version 4.3; Silk Scientific, Orem, UT).

Statistical analyses. The data in all graphs are presented as means ± SE. Statistical analyses (StatView, Abacus Concepts, Berkeley, CA, 1992) consisted of one-sample t-test or one- or two-factor univariate ANOVA. When appropriate, post hoc comparisons between individual group means were made by Fishers protected least significant differences tests. A P value of <0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The expression of HIF-1{alpha} and -2{alpha} proteins for all subjects is shown in Fig. 1. Figure 1A portrays a representative Western blot. Because multiple gels were required to assess placental HIF-{alpha} protein levels for all patients in each group (a total of 50 subjects), we made a ratio of HIF-{alpha} expression between each of the abnormal placentas and a normal placenta loaded adjacently in the gel. (The normal and pathological placentas were randomly matched.) Thus, a ratio equal to 1.0 indicates no difference in HIF-{alpha} expression between the normal-term and abnormal placentas. In this way, the assay variation among Western blots was circumvented, thereby allowing for compilation of the data as shown in Fig. 1B. Both HIF-1{alpha} and -2{alpha} levels were significantly increased in preeclamptic relative to normal-term placentas, the means ± SE of ratios being 1.67 ± 0.15 and 1.72 ± 0.20, respectively (P < 0.001 and < 0.01 vs. 1.0). In contrast, there was no significant elevation of HIF-1{alpha} or -2{alpha} in the placentas from normotensive women delivering growth-restricted babies, the ratios being 1.03 ± 0.07 and 0.96 ± 0.16, respectively (both P = not significant vs. 1.0).


Figure 1
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  		Fig. 1. Western blot analyses of hypoxia-inducible transcription factor-1{alpha} (HIF-1{alpha}) and -2{alpha} proteins in placental biopsies obtained from women with normal (NP) or preeclamptic (PE) pregnancies, or from normotensive pregnant women who delivered growth-restricted babies [intrauterine growth restriction (IUGR), < 10% birth weight, n = 10] without PE. Pregnancies resulting in both normally grown (>10%, n = 13) and small babies (≤10%, n = 5) are included in the PE group. A: representative Western blot. B: HIF-{alpha} protein in each abnormal placenta is expressed as the ratio of HIF-{alpha} protein in a normal-term placenta. The individual ratios for n = 18 placental pairs (each for PE/NP) and n = 10 placental pairs (each for IUGR/NP) are averaged and depicted in the graph. A stippled line indicates a ratio of 1.0. If the ratio is not significantly different from unity, then expression of HIF-{alpha} protein is comparable in the placentas from abnormal and normal pregnancies. **P ≤ 0.001 and *P < 0.01 vs. 1.0.

 
Because the results in Fig. 1 included all women with preeclampsia, we further analyzed the data according to baby weights > 10% (n = 13 patients) and ≤ 10% (in fact, ≤5.5%, n = 5; Fig. 2A). The increase in HIF-{alpha} was comparable between these two groups of preeclamptic women.


Figure 2
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  		Fig. 2. Additional analyses of HIF-1{alpha} and -2{alpha} proteins in placental biopsies. HIF-{alpha} protein in each abnormal placenta is expressed as the ratio of HIF-{alpha} protein in a normal-term placenta. A: HIF-{alpha} protein levels in n = 5 placentas from women with PE who delivered growth-restricted babies of <10% birth weight (in fact, ≤5.5%), and n = 13 placentas from women with PE who delivered babies >10% birth weight expressed as a ratio of HIF-{alpha} protein in normal-term placentas. B: HIF-{alpha} protein levels are portrayed for placentas from normotensive women delivering growth-restricted babies ≤2.4% and >2.4%, n = 7 each. **P < 0.01, *P < 0.05, and {dagger}P < 0.1 vs. 1.0.

 
We further analyzed the data for the normotensive IUGR pregnancies shown in Fig. 1 by comparing those delivering babies of ≤2.4% (n = 7 patients) vs. >2.4% (n = 7; Fig. 2B). The former are more likely to have abnormal trophoblast invasion and absent remodeling of spiral arteries, which is believed to compromise placental perfusion and delivery of oxygen and nutrients (13). Once again, however, neither HIF-1{alpha} nor -2{alpha} protein was significantly increased in placentas from women delivering asymmetrically grown, small babies.

The expression of placental Flt-1 and sFlt-1 proteins for all subjects is shown in Fig. 3. Figure 3A portrays a representative Western blot. Again, because multiple gels were required to assess placental Flt-1 proteins for all patients in each group, we made a ratio of Flt-1 or sFlt-1 expression between each abnormal and normal placenta. Thus a ratio equal to 1.0 indicates no difference in the expression of Flt-1 proteins between normal and abnormal placentas. In this way, the assay variation among Western blots was circumvented, thereby allowing for compilation of the data as shown in Fig. 3B. Both Flt-1 and sFlt-1 levels were significantly increased in preeclamptic relative to normal-term placentas, the means ± SE of ratios being 2.23 ± 0.30 and 2.11 ± 0.40, respectively (both P < 0.001 vs. 1.0). In contrast, there was no significant elevation of Flt-1 or sFlt-1 in the placentas from women delivering asymmetrically grown, small babies without preeclampsia, the ratios being 1.14 ± 0.15 and 0.95 ± 0.12, respectively (both P = not significant vs. 1.0).


Figure 3
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  		Fig. 3. Western blot analysis of HIF-{alpha}-regulated proteins, VEGF receptor, Flt-1, and intracellular soluble fms-like tyrosine kinase 1 (sFlt-1), in placental biopsies obtained from women with NP or PE pregnancies, or from normotensive pregnant women who delivered growth-restricted babies (IUGR; <10% birth weight, n = 10) without PE. Pregnancies resulting in both normally grown (>10%, n = 13) and small babies (≤10%, n = 5) are included in the PE group. A: representative Western blot. B: Flt protein in each abnormal placenta is expressed as the ratio of Flt protein in a normal-term placenta. The individual ratios for n = 18 placental pairs each for PE/NP and n = 10 placental pairs for IUGR/NP are averaged and depicted in the graph. The stippled line indicates a ratio of 1.0. If the ratio is not significantly different from unity, then expression of Flt protein is comparable in the placentas from abnormal and normal pregnancies. **P ≤ 0.001 vs. 1.0.

 
Because the results in Fig. 3 included all women with preeclampsia, we further analyzed the data according to baby weights of >10% (n = 13 patients) and ≤10% (in fact, ≤5.5%, n = 5; Fig. 4A). The increases in Flt-1 and sFlt-1 were comparable between these two groups of preeclamptic women.


Figure 4
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  		Fig. 4. Additional analyses of Flt proteins in placental biopsies. Flt protein in each abnormal placenta is expressed as the ratio of Flt protein in a normal-term placenta. A: Flt-1 and sFlt-1 protein levels in n = 5 placentas from women with PE who delivered growth-restricted babies of <10% birth weight (in fact, ≤5.5%) and n = 13 placentas from women with PE who delivered babies >10% expressed as a ratio of HIF-{alpha} protein in normal-term placentas. B: Flt-1 and sFlt-1 protein levels are portrayed for placentas from normotensive women delivering growth-restricted babies ≤2.4% and >2.4%, n = 7 each. **P < 0.01 and {dagger}P < 0.1 vs. 1.0.

 
We further analyzed the data in Fig. 3 for the normotensive IUGR pregnancies by comparing those delivering babies of ≤2.4% (n = 7 patients) vs. >2.4% (n = 7; Fig. 4B). Again, the former are more likely to have abnormal trophoblast invasion and absent remodeling of spiral arteries (13). However, neither Flt-1 nor sFlt-1 protein was significantly increased in placentas from women delivering asymmetrically grown, small babies.

One possible explanation for the lack of increase of HIF-{alpha} protein expression in placentas from normotensive women delivering growth-restricted babies is that there is inadequate HIF-{alpha} mRNA to permit HIF-{alpha} protein accumulation despite protein stabilization under hypoxia (36). Figure 5A depicts a representative Northern blot. After normalizing for 18S RNA, we made a ratio of HIF-1{alpha} or HIF-2{alpha} mRNA expression between each abnormal and normal placenta. A compilation of the data is shown in Fig. 5B; HIF-1{alpha} and -2{alpha} mRNA levels were comparable among the normal and abnormal placentas, the ratios not being significantly different from 1.0.


Figure 5
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  		Fig. 5. Northern blot analysis of HIF-1{alpha} and -2{alpha} mRNA in placental biopsies obtained from women with NP or PE pregnancies or from normotensive pregnant women who delivered growth-restricted babies (IUGR, <10% birth weight) without PE. Pregnancies resulting in both normally grown (>10%, n = 5) and small babies (≤10%, n = 3) are included in the PE group. A: representative Northern blot for HIF-1{alpha} and -2{alpha} is depicted. B: densitometry was performed and HIF-{alpha} values were first normalized to 18S RNA. Then, normalized HIF-{alpha} mRNA in each abnormal placenta is expressed as the ratio of HIF-{alpha} mRNA in a normal-term placenta. The individual ratios for n = 8 placental pairs each for PE/NP and IUGR/NP are averaged and depicted in the graph. The stippled line indicates a ratio of 1.0. If the ratio is not significantly different from unity, then expression of HIF-{alpha} mRNA is comparable in the placentas from abnormal and normal pregnancies.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major and unexpected finding of this work is that HIF-{alpha} proteins are not increased in placentas obtained from normotensive pregnant women who delivered small, asymmetrically grown babies of ≥37 wk of gestation. These results may be particularly reliable for several reasons. First, we studied placentas from 10 growth-restricted pregnancies that were clinically well-defined. Second, we concurrently investigated two HIF-{alpha}-regulated genes, Flt-1 and sFlt-1 (38), in the same placental homogenates, and like HIF-{alpha}, they were not increased in the placentas from the growth-restricted pregnancies. Third, as a positive control, we simultaneously studied placentas from 18 women with rigorously diagnosed preeclampsia, and as previously reported (27, 29), HIF-{alpha} and Flt proteins were significantly increased by ~50–100% relative to normal-term placentas (and preterm placentas) (29).

In historical context, Dr. Page first advanced the concept of a "placental pressor substance capable of being produced by all chorionic tissue in response to a relative ischemia" (25). Placental intervillous space PO2 is determined by the balance of three factors: uteroplacental oxygen delivery to the placenta, placental oxygen consumption (as much as 40% of total uterine oxygen consumption) (8), and fetal oxygen extraction from the placenta.

There is considerable indirect evidence for placental ischemia-hypoxia in preeclampsia (reviewed in Ref. 10): 1) reduced uteroplacental blood flow suggested by ultrasonography, as well as by older techniques using clearance rates of various radioactive compounds and steroids; 2) 30% incidence of IUGR; 3) increased frequency of placental infarcts relative to normal placentas; 4) altered placental morphology including increased terminal villous capillary branching, vasculosyncytial membrane formation, and villous cytotrophoblast proliferation; and 5) preeclampsia-like syndrome produced by uteroplacental ischemia in animal models.

There is also correlative evidence (see Ref. 10, and citations therein): 1) increased incidence of disease in women residing at high altitude; 2) increased incidence in women with preexisting vascular compromise that may involve uterine arteries, e.g., hypertension and diabetes mellitus; 3) increased incidence in women with large placental mass, e.g., twin gestation and hydatiform mole; and 4) salutary effect of bed rest and left lateral recumbency, which improve uteroplacental perfusion.

We now have molecular evidence of placental ischemia-hypoxia in preeclampsia. Both HIF-1{alpha} and -2{alpha}, the master regulators of the cellular response to hypoxia, are increased in preeclamptic placentas (Refs. 27 and 29 and present study). In addition, downstream genes that are known to be regulated by HIF-{alpha} are increased in the placentas of preeclamptic women (Refs. 1, 7, 17, 19, 22, 24, 35, and 37 and present study): TGF-beta3, teleomerase reverse transcriptase, LDH-A4, tyrosine hydroxylase, Flt-1, sFlt-1, endoglin and soluble endoglin, and pyroyl hydroxylase-3. Finally, DNA microarray and suppressive-subtractive hybridization analyses demonstrate global increase in hypoxia-activated genes (33, 36).

Nevertheless, there is one caveat. HIF-{alpha} protein abundance, transactivational activity, or both can also be stimulated under nonhypoxic conditions, e.g., by growth factors and cytokines, such as insulin growth factor-1 and angiotensin II, and by small molecules, such as nitric oxide and reactive oxygen species, as well as other factors (38). However, the mechanism of stimulation may be different, e.g., IGF-1 and angiotensin II increase HIF-{alpha} protein abundance through increasing translational efficiency rather than protein stabilization as occurs under hypoxia (38). On balance, after considering all of the evidence reviewed above (indirect, correlative, and molecular), uteroplacental oxygen delivery in preeclampsia is likely to be reduced relative to placental and/or fetal oxygen consumption, thereby rendering a relatively hypoxic intervillous space and placenta.

Interestingly, increased expression of HIF-{alpha} and downstream genes Flt-1 and sFlt-1 were comparable in placentas from preeclamptic women with normally grown or growth-restricted fetuses. Because uterine blood flow is believed to be more severely compromised in the latter, we anticipated even higher levels of HIF-{alpha} and regulated genes. On a cautionary note, however, only five of 18 or 30% of the preeclamptic pregnancies were associated with fetal growth restriction in agreement with the literature (20). This low number of cases may have precluded the detection of any further increase in the expression of HIF-{alpha} and regulated genes in this subgroup of preeclamptic subjects. Alternatively, restricted fetal and placental growth may reduce oxygen consumption, thereby offsetting the more severely compromised uterine blood flow and oxygen delivery and preventing further increases in HIF-{alpha} and regulated genes.

Of additional note is the lack of association between gestational age and HIF-1{alpha} and -2{alpha}, Flt-1 and sFlt-1 protein expression in placentas from preeclamptic women as shown in Fig. 6, which is a compilation of individual data from the present and our previous studies (27, 29). On the one hand, we expected that early, severe preeclampsia might be associated with higher levels of placental HIF-{alpha} and regulated genes, due to more severely compromised trophoblast invasion, spiral artery remodeling, uterine blood flow, and oxygen delivery. On the other hand, early, severe preeclampsia is frequently associated with both abnormal uterine and umbilical artery Doppler waveforms (12), the latter suggesting reduced fetal oxygen extraction, which may mitigate further increases in HIF-{alpha}. Unfortunately, we did not obtain placentas from women with early-onset IUGR (without preeclampsia) to make a similar correlation (see below).


Figure 6
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  		Fig. 6. Compilation of HIF-1{alpha} (A), HIF-2{alpha} (B), Flt-1 (C), and sFlt (D) protein levels in placentas from PE women expressed as a function of gestational age and as the ratio of their respective proteins in a normal-term placenta (from the present study and Refs. 27, 29). Each point represents one placental pair. There was no significant correlation between HIF-{alpha} or Flt proteins and gestational age.

 
The main goal of this study was to investigate the status of placental HIF-{alpha} expression in normotensive women who delivered growth-restricted babies, because both preeclampsia and IUGR are thought to share a common placental etiology, namely, shallow placentation and placental hypoxia (21). Indeed, Mayhew et al. (18) stated that "...late onset IUGR and presence of end diastolic waveforms on umbilical artery Doppler ultrasound belong to the uteroplacental type of hypoxia since [it is] associated with ischemia-hypoxia in which delivery of blood flow to the intervillous space is compromised by events initiated early in pregnancy when there is deficient trophoblast invasion of spiral arteries." Thus, we studied placentas from normotensive women who delivered asymmetrically grown babies of ≥37 wk of gestation with normal umbilical artery Doppler waveforms. However, the data show that HIF-1{alpha} and -2{alpha} proteins, as well as the downstream genes sFlt-1 and Flt-1 are not increased in these placentas even from pregnancies that resulted in a baby of ≤2.4%, which has been shown to be associated with absent trophoblast invasion and spiral artery remodeling (13). In future studies, it would be worthwhile to concurrently perform placental bed biopsies to document the degree of deficient trophoblast invasion and spiral artery remodeling and measurements of molecular markers of villous oxygenation. Indeed, one possibility is that trophoblast invasion and spiral artery remodeling are not compromised in normotensive patients with IUGR babies of >37 wk gestation with birth weights of <10% (or ≤2.4% for that matter, see above) and asymmetrical growth.

A previous investigation suggested a global decrease in transcriptional activity and mRNA abundance in placentas from IUGR pregnancies (36). However, absence of HIF-{alpha} induction in our study was not a consequence of insufficient mRNA to make protein for stabilization by hypoxia as assessed by Northern blot analysis. Comparable levels of HIF-{alpha} mRNA were observed between normal-term placentas and placentas from normotensive IUGR and preeclamptic women, the latter corroborating earlier work (29). Another explanation is that the IUGR placentas are unable to sense or respond to hypoxia, a possibility to be tested in the future.

The most likely explanation for the present findings is that, in contrast to preeclampsia, uteroplacental oxygen delivery is matched to reduced placental and/or fetal oxygen consumption such that the intervillous/placental PO2 is relatively nonhypoxic (26). This conclusion is consistent with the finding that sFlt-1 is not increased in the maternal circulation of these women (12, 32). It is also consistent with one (36) but not another (31) report on the status of hypoxia-activated genes in placentas from IUGR pregnancies as determined by subtractive-suppressive hybridization and DNA microarray, respectively.

The present results raise a conundrum. In late-onset IUGR, does reduced uteroplacental oxygen delivery limit fetal oxygen extraction and hence growth, or does reduced fetal oxygen extraction and growth limit uteroplacental blood flow? The latter suggests that some cases of late-onset IUGR may represent a primary reduction in fetal oxygen extraction with appropriately matched (reduced) placental growth, trophoblast invasion, spiral artery remodeling, and uteroplacental blood flow.

We speculate that the potential link between fetal growth and uteroplacental blood flow may be intervillous space/placental PO2. In normal pregnancy, fetal growth increases fetoplacental oxygen extraction, thereby reducing intervillous space/placental PO2. [Note that HIF-{alpha} expression is exquisitely sensitive to small changes of PO2 in the physiological range (28, 38)]. The reduced PO2 in turn stimulates expression of placental HIF-{alpha} and downstream genes, such as vascular endothelial and other growth factors, the latter serving to increase placental size and vascularization, and perhaps through venoarterial exchange (9) to enhance uterine blood flow by promoting trophoblast migration and invasion on the one hand and by dilating spiral and more proximal uterine arteries on the other. The opposite chain of events may pertain to some cases of late IUGR. With reduced fetal growth, fetoplacental oxygen extraction is less (or vice versa, see below), such that an imbalance between uteroplacental oxygen delivery and consumption is not produced, and intervillous space/placental PO2 is relatively nonhypoxic. In the absence of reduced PO2, HIF-{alpha} and regulated genes are not induced, thereby depriving (and appropriately so) the placenta of growth factors to grow and vascularize, and the placental bed of these signals to promote trophoblast migration and invasion and dilation of uterine arteries. Thus, the lack of placental growth, trophoblast invasion, spiral artery remodeling, and dilation is a secondary rather than a primary event. Because the placenta virtually doubles in diameter from 20 gestational weeks to term (3), trophoblast invasion and spiral artery remodeling likely occur throughout gestation, and therefore, these processes could conceivably be modified (restrained) as described above by fetal growth restriction occurring later in pregnancy. (Note, however, that despite its logical basis, to our knowledge there are no data to support the contention that trophoblast invasion and remodeling entails progressively more lateral arteries as gestation advances.)

In addition to a primary reduction in fetal growth, another potential explanation for reduced fetoplacental oxygen extraction is a primary increase in fetoplacental vascular resistance and/or decreased compliance (due to abnormal arterial function and structure or inadequate vascularity) albeit typically below the sensitivity of umbilical artery Doppler ultrasound as applied clinically (Table 3), perhaps as a consequence of epigenetic or genetic inheritance. This concept of fetoplacental vascular abnormalities in utero that are inherited may be another explanation for the apparent predisposition of some babies who are born small to develop cardiovascular disease in adult life (2).

Although we did not have placentas from early IUGR available to us, there are relevant data emerging from other labs that need mention here. Traditionally, early IUGR with abnormal umbilical artery Doppler waveforms is considered to be postplacental hypoxia, although to our knowledge, HIF-{alpha} protein expression has not been reported for these placentas. Thus, the PO2 of the intervillous space and villous placenta has been considered to be nonhypoxic or even relatively hyperoxic (3). However, recent evidence suggests increased sFlt-1 expression in the placenta (23, 34), as well as increased maternal circulating levels of sFlt-1 in these pregnancies (12). Insofar as sFlt-1 is regulated by hypoxia and HIF-{alpha}, these findings do not support the concept of postplacental hypoxia in early IUGR. However, an explanation that may reconcile this apparent contradiction is that, if HIF-{alpha} is found to be increased, it may be elevated on the basis of excess reactive oxygen species, which can stabilize HIF-{alpha} protein by any one of several mechanisms impairing degradation (38).

A noninvasive measurement of the relative state of intervillous space/placental oxygenation would be useful in the prediction, classification, and treatment of the obstetrical diseases believed to be related to inadequate placentation (i.e., IUGR, preeclampsia, and preterm labor). In this regard, noninvasive approaches, such as near-infrared spectroscopy and blood oxygen level-dependent magnetic resonance imaging may prove to be safe and reliable methodologies (15, 16). When relative placental ischemia-hypoxia is a component of the disease, then strategies to improve uteroplacental blood flow may be beneficial, to improve placental and fetal growth, and/or alleviate maternal endothelial dysfunction by reducing circulating toxic factors that emanate from the placenta as a consequence of local hypoxia. In this regard, recombinant human relaxin may be efficacious by not only improving maternal renal and systemic hemodynamics (without causing undue systemic hypotension) but also by vasodilating unremodeled spiral arteries and more proximal, vasoconstricted uterine arteries (11, 14).


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Child Health and Human Development Grant PO1-HD-30367. Dr. Jeyabalan is supported by the Building Interdisciplinary Research Career in Women's Health Faculty Development Award (National Institute of Child Health and Human Development Grant K12-HD-043441).


   ACKNOWLEDGMENTS
 
Portions of this work were published in abstract form in J Soc Gynecol Invest 13: 229A, 2006.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. P. Conrad, Dept. of Physiology and Functional Genomics, Univ. of Florida College of Medicine, 1600 SW Archer Road, M552, PO Box 100274, Gainesville, FL 32610-0274 (e-mail: kpconrad{at}ufl.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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