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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Placenta. Author manuscript; available in PMC Oct 1, 2010.
Published in final edited form as:
PMCID: PMC2771543
NIHMSID: NIHMS138362
EFFECT OF HEME OXYGENASE-1 DEFICIENCY ON PLACENTAL DEVELOPMENT
Hui Zhao,a Ronald J. Wong,a Flora S. Kalish,a Nihar R. Nayak,b and David K. Stevensona
aDept. of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305
bDept. of Obstetrics & Gynecology, Stanford University School of Medicine, Stanford, CA 94305
Corresponding author: Hui Zhao, Dept. of Pediatrics, Stanford University School of Medicine, 300 Pasteur Drive, Rm S230, Stanford, CA 94305-5208, huizhao2/at/stanford.edu; Ph: 650-498-7246; Fax: 650-725-7724
Heme oxygenase (HO) is the rate-limiting enzyme in the heme catabolic pathway and highly expressed in the placenta. Deficiencies in HO-1, the inducible isoform, have been associated with pregnancy disorders, such as recurrent miscarriages, intrauterine growth retardation, and pre-eclampsia. The aim of this study was to identify if a deficiency in HO-1 affects placental development using a mouse model. When HO-1 heterozygote (Het, HO-1+/−) mice were cross-bred, an extremely low birth rate in homozygote (Mut, HO-1−/−) offspring (2.4%) and small litter sizes were observed. Placentas and fetuses from Het cross-breedings were relatively smaller and weighed less than those from wild-type (WT) cross-breedings at E12.5 and E15.5. Furthermore, Het placentas had significantly less HO-1 mRNA and protein levels than WT placentas, but no significant differences in placental HO activity. Interestingly, HO-2, the constitutive HO isoform, as well as iNOS and eNOS expression were significantly upregulated in Het placentas. Histological examination showed that the junctional zone (JZ) of Het placentas were markedly thinner than those of WT placentas and appeared to be due to an increase in apoptosis. Immunohistochemistry revealed that HO-1-expressing cells were located primarily in the JZ of Het placentas, specifically in the spongiotrophoblast layer. In addition, diastolic blood pressures and plasma soluble VEGFR-1 (sFlt-1) levels were significantly elevated in pregnant Het mice. We conclude that a partial deficiency in HO-1 is associated with morphological changes in the placenta and elevations in maternal diastolic blood pressure and plasma sFlt-1 levels, despite a compensatory increase in HO-2 expression.
Heme oxygenase (HO) is the rate-limiting enzyme that degrades heme to produce equimolar quantities of carbon monoxide (CO), iron, and biliverdin, which is immediately reduced to bilirubin [1]. Two primary HO isoforms have been identified in the human and rodent: the inducible HO-1 and the constitutive HO-2 [24]. Expression of HO-1, also known as Hsp32, can be induced by many factors, e.g. heavy metals, oxidative stress, UV radiation, lipopolysaccharide (LPS), thermostress, etc. [2]. Besides maintaining homeostasis through the regulation of cellular heme and hemoprotein levels [5], HO-1 also possesses anti-inflammatory, antioxidant, and anti-apoptotic properties [6,7], mediated partly by the production of the bioactive metabolites, CO and biliverdin/bilirubin [5,8,9].
The mammalian placenta is a vital organ in pregnancy, acting as the interface between maternal and fetal environments to provide essential nutrients and regulate maternofetal hemodynamics. Many pregnancy disorders are due to poor placentation, improper placental hemodynamic regulation, and insufficient antioxidant protection. In placentas from patients with pregnancy complications, a down-modulation or reduction in HO-1 expression, was associated with recurrent miscarriages, spontaneous abortions, and pre-eclampsia [1013]. End-tidal breath CO levels were found to be significantly lower in women with pregnancy-induced hypertension compared to controls [14,15]. In humans, there are interindividual variations in HO-1 expression, mainly due to 2 genetic phenotypes: (1) polymorphisms of (GT)n dinucleotide repeats in the HO-1 regulatory region [longer repeats are associated with decreased gene expression] with an increased size of the (GT)n associated with idiopathic recurrent miscarriage [11]; and (2) HO-1 mutant alleles. The first identified human case of HO-1 deficiency was a 6-yr-old boy, who had both alleles encoding a truncated HO-1 protein. He had marked growth retardation and developmental delay and eventually died [16]. His mother, a heterozygous carrier, had 2 previous intrauterine fetal deaths, suggesting a key role of HO-1 in maintaining a healthy pregnancy.
The physiological importance of HO-1 in pregnancy has also been reported in the full HO-1 knock-out (Mut, HO-1−/−) mouse [17]. Mut:Mut matings did not yield viable litters and that heterozygous- (Het, HO+/−):Het matings did not result in the expected Mendelian ratio – only 5% were Mut compared to the expected 25%. Along with a low survival rate, Mut offspring were smaller than wild-type (WT) and Het littermates and remained so through early adulthood [17].
Expression and localization of HO-1 protein in the murine placenta have been characterized [18,19]. As early as E6.5, HO-1 is expressed in the ectoplacental cones. After placental maturation (E14.5 in mouse and E15.5 in rat), HO-1 is restricted to trophoblast cells, specifically in the spongiotrophoblast (SP) layer in the junctional zone (JZ) [18]. Upregulation of HO-1 in the placenta and uterus facilitates tissue-protective functions and promotes graft acceptance of allogeneic fetuses. Zenclussen et al [20] reported that HO-1 and HO-2 were significantly downregulated in placental tissues from allopregnant mice undergoing abortions. Pregnancy outcomes improved with the overexpression of placental HO-1 with a related upregulation of Bag-1, a tissue protective marker, and neuropilin-1, a receptor activating T regulatory cells [21,22].
We have previously shown that HO and CO function as vasodilators and are important for the maintenance of maternal vascular tone and the regulation of materno- and placentofetal hemodynamic function [23]. Inhibition of placental HO by the inhibitor, tin mesoporphyrin (SnMP), significantly increased maternal blood pressure [23]. HO-1 can also regulate placental angiogenesis through both VEGF and sFlt-1 (soluble VEGFR-1) [24,25]. sFlt-1 is the primarily form of Flt-1 produced in the mouse and human placenta during the later stages of gestation. Women with pre-eclampsia have elevated serum sFlt-1 and soluble endoglin levels [24,26,27]. Cudmore et al [25] have shown that HO-1 can negatively regulates sFlt-1 and soluble endoglin in vitro, and that the administration of CO-releasing molecules or endogenous CO can decrease sFlt-1 release.
In this present study, we investigated if HO-1 plays a role in placental development and in the maintenance of a healthy pregnancy using a mouse model of HO-1 deficiency.
2.1. Animals
C57BL/6 HO-1/KO mice [17] were backcrossed with FVB mice to produce an FVB HO-1/KO (Mut) mouse line. Mice were maintained under strict adherence to Stanford University guidelines and mated at 6–8 wks of age. Gestational age was determined by vaginal plug day (E0.5) and confirmed by embryonic length using micro-ultrasound.
2.2. Mouse genotyping
Genomic DNA was isolated from tail clippings or fetal tissues using the Tissue DNeasy kit (Qiagen, Germany) and typed by PCR. For HO-1+/+, HO-1+/−, and HO-1−/− screening, 2 sets of primers designed for the WT, Het, and Mut were used. Conditions for PCR were as follows: 95°C for 10 min for denaturing the genomic DNA, 94°C for 20 sec, 68°C for 30 sec, and 72°C for 40 sec, repeating 40 cycles. WT (510 bp) and Het and Mut (390 bp) bands were analyzed using electrophoresis.
2.3. HO activity measurements
Tissue HO activity was determined by gas chromatography (GC) [28]. Briefly, mouse placentas were harvested and sonicated (10% w/v) in phosphate buffer (pH 7.4). Sonicates were then incubated with equal (20 µL) volumes of NADPH (4.5 µM) and methemalbumin (50 µM/11.2 µM) for 15 min at 37°C in 2-mL CO-purged vials. CO released into the headspace was determined by GC. HO activity was expressed nmol CO/h/g fresh weight (FW).
2.4. Western blot
100 µg of sonicates were boiled for 10 min in protein loading buffer and separated by electrophoresis. Proteins were transferred to PDVF membranes (Bio-Rad, Hercules CA) using a semidry transblotter (Bio-Rad). Membranes were probed with polyclonal antibodies raised against HO-1, HO-2 (Stressgen Corp., Victoria, CN), and Bag-1 (Santa Cruz Biotechnology, Santa Cruz CA). Protein levels were quantitated by densitometry. Coomassie blue staining or β-actin immunoblotting were performed to confirm equal loading of samples.
2.5. Quantitative RT-PCR analysis
After sacrifice, placentas were immediately stored in RNAlater (Qiagen). Total RNA was extracted using the RNAeasy Mini Kit (Qiagen). HO-1, HO-2, iNOS, eNOS, P21, Mash-2, neuroplin-1 (Nrp-1), and β-actin were quantified using the Quanti-Tect SYBR Green RT-PCR kit (Qiagen). Amplification was performed using an Mx-3005 TM Quantitative PCR Systems (Stratagene, Cedar Creek TX) [23,29].
2.6. Histological staining
Placentas were collected and placed in Protocol* 10% Neutral Buffered Formalin (Fisher Scientific). Fixed placentas were embedded in paraffin according to standard protocols. 6-µm thick tissues were sectioned using a microtome. Following deparaffinization, sections were stained with H&E (American Master*Tech Scientific, Lodi CA) and placental structures visualized by light microscopy.
2.7. HO-1 and HO-2 immunostaining
HO-1- and HO-2-expressing cells were identified by immunohistochemistry using the Dako EnVision system (DakoCytomation, Carpinteria CA). Briefly, 6-µm thick sections were cut from paraffin-embedded tissues. After deparaffinization, sections were treated using the Antigen Retrieval Citra Plus solution (BioGenex, San Ramon CA), followed by an endogenous peroxidase solution, and then incubated with primary antibodies against HO-1 and HO-2 (Stressgen Corp). Bound antibodies were detected using a labeled polymer and colorized with the DAB+ substrate chromogen solution. Negative controls were run in parallel using adjacent placental sections incubated with a non-specific antibody (DakoCytomation). Presence of HO-1 or HO-2 was indicated by a brown or black color within the cytoplasm, respectively. Counterstaining was performed with a hematoxylin solution.
2.8. TUNEL assay
Paraffin-embedded placental tissues were sectioned to 5 µM, de-paraffinized by xylene, and rehydrated in ethanol. TUNEL staining was performed using the In Situ Cell Death Detection Kit, TMR red (Roche, Germany). Tissue sections incubated with labeling solution only (i.e., without enzyme) served as negative controls. Samples first treated with recombinant DNaseI (RNase-free) followed by the standard protocol serves as positive controls. Extent of apoptosis was expressed as the total number of TUNEL-stained cells counted under 200X magnification.
2.9. Prussian blue iron staining
Ferric iron and ferritin deposition in placentas were detected using the Prussian Blue Reaction Kit (Polysciences, Warrington PA). Iron-positive cells stained bright blue, while nuclei and cytoplasm stained pink to red following nuclear fast red counterstaining.
2.10. Uterine NK cell staining
Dolichos biflorus agglutinin (DBA) staining was used to identify uNK cells. Briefly, placental sections were de-paraffinized in xylene, re-hydrated in ethanol, and then treated with 1% H2O2 for 30 min to block endogenous peroxidase activity. Samples were incubated for 30 min in 1% BSA followed by biotinylated-DBA lectin (1:400, Vector Labs, Burlingame CA) at 37°C for 2h. Sections were washed and incubated for another 30 min at 20°C with RTU Vectastain ABC peroxidase-conjugated streptavidin (Vector Labs). uNK cells were identified using the Liquid DAB+ substrate chromogen solution (Vector Labs) with positive cells staining brown. Sections were further counterstained with Harris’ hematoxylin and mounted with an aqueous mounting medium.
2.11. Blood pressure measurements
Blood pressures were monitored daily using a tail cuff system (BP-2000, Visitech System, Apex NC). Mice were trained for 7 days prior to recording all values. The apparatus was calibrated to inflate from 50–200 mm Hg. Mean±SD systolic and diastolic blood pressure values were calculated from 20 measurements per mouse.
2.12. sFlt-1 measurements
Blood was collected by retro-orbital bleeding, transferred to heparin-filled gel tubes (CapiJect, Terumo Medical Corp., Somerset NJ), and centrifuged at 13,000xg for 1 min to separate plasma. sFlt-1 levels were then measured using a mouse VEGF/sFlt-1 ELISA development kit (R&D Systems, Minneapolis MN). Samples were diluted 1:10 with dilution buffer. Standard curves, ranging from 125–8000 pg/mL were prepared for sFlt-1. Plates were read at 450 nm using a VersaMax tunable microplate reader.
2.13. Statistical analysis
For all comparisons between experimental groups, paired or unpaired t-tests were performed for each set of experiments to determine statistically significant differences at p≤0.05.
3.1. HO-1 deficiency results in fetal lethality and decreased litter sizes
HO-1+/− mice were cross-bred and offspring phenotype and litter sizes recorded. 432 embryos/pups (~84 mating) were screened. Phenotypic distribution of the offspring were as follows: 25.5% HO-1+/+, 72.1% HO-1+/−, and only 2.4% HO-1−/−, a percentage less than the predicted 25%. When we compared the average pup number per litter, HO-1+/− breedings produced ~5.1 pups/litter, which was significantly lower than ~9.2 pups/litter from WT matings. The low birth rate of HO-1−/− offspring and small litter sizes suggest that they are mainly due to intrauterine abortions, which we have observed regularly in HO-1+/− cross-breedings. Most Mut embryos aborted before E10.5.
3.2. Developmental delay in HO+/− placentas and fetuses
To investigate if a partial deficiency in HO-1 results in placental and fetal growth retardation, placentas and fetuses from HO-1+/+ (yielding embryos w/WT) and HO-1+/− cross-breedings (yielding embryos h/WT, h/Het, and h/Mut) were collected at E12.5, E15.5, and E18.5 and weighed (Table 1). Placentas and embryos from HO-1+/− pregnancies were lighter and smaller than those from WT pairings. Moreover, h/Mut offspring were smaller than h/Het offspring, which were smaller than h/WT offspring. Significant differences were found at E12.5 and E15.5, however, these discrepancies diminished as delivery approached (E18.5).
Table 1
Table 1
Developmental Delay of Placentas and Fetuses in HO-1-Deficient Pregnancies.
3.3. HO-1 and HO-2 mRNA and HO activity levels in the developing HO+/− placentas
To investigate if HO-1 expression was reduced in HO-1 deficient placentas, HO-1 mRNA levels were measured (Fig. 1A). h/Mut placentas were not analyzed in these experiments due to insufficient Mut samples. In WT breedings, placental HO-1 mRNA levels peaked at ~E15.5. w/WT placentas had the highest mRNA levels followed by those of h/WT. Compared to h/WT placentas, HO-1 mRNA levels in h/Het placentas were significantly reduced by 22% at E12.5 (p<0.05), by 51% at E15.5 (p<0.05), and by 47% at E18.5 (p<0.05). In contrast, HO-2 mRNA levels in h/Het placentas were significantly increased by 17% and 21% (p<0.05) at E15.5 only compared to w/WT and h/Het placentas, respectively (Fig. 1B).
Fig. 1
Fig. 1
HO-1 and HO-2 expression and total HO activity levels in developing placentas. Placentas from w/WT (black, n=3), h/WT (dark grey, n =4), and h/Het (light grey, n =4) pregnancies were collected at various gestational ages. (A) HO-1 and (B) HO-2 mRNA levels (more ...)
When total placental HO activity from w/WT, h/WT, and h/Het embryos were determined at E13.5, E15.5, and E17.5 (Fig. 1C), HO activity was greatest at ~E15.5 for all genotypes. No significant differences in HO activity were found in placentas from w/WT and h/WT embryos at all ages. However, h/Het placentas showed ~10% less activity than those WT and Het/WT embryos, with significance observed at E17.5 (p<0.05).
3.4. Upregulation of HO-2 protein in HO+/− placentas
To investigate if there was a compensatory increase in HO-2 expression in HO-1 deficiency, HO-1 and HO-2 protein levels were measured in placentas harvested at E11.5–E15.5. Compared to placentas from h/WT embryos, HO-1 protein in h/Het placentas were significantly lower by 20%–50% (Fig. 1D), while HO-2 levels increased by 25%–70% (Fig. 1E).
3.5. Structural changes in the SP layer in HO+/− placentas
When placentas from w/WT, h/WT, and h/Het embryos were compared at E14.5, the SP layers of h/Het placentas (Fig. 2B and 2D) were found to be markedly thinner and disorganized, with borders less well-defined, than those of w/WT placentas (Figs. 2A and 2C). In contrast, h/WT placentas had relatively thicker SP layers than those from h/Het embryos, but the borders were not well connected (data not shown). Also, islands of SP-like cells were scattered within the labyrinth regions of h/Het placentas, which was not observed in w/WT placentas. Immunohistochemical staining for HO-1 (Figs. 2E and 2F) showed that the intense HO-1 signals were found in the JZ, especially in the SP layer. The HO-1-positive regions of h/Het placentas were much thinner than that of w/WT placentas (controls). However, by E18.5, no significant morphological differences were observed between any genotype (data not shown).
Fig. 2
Fig. 2
Structural changes of the SP layer in HO-1+/− placentas. Representative of microscopic images of H&E-stained placentas from w/WT (A and C, 40X and 200X, respectively) and h/Het (B and D, 40X and 200X, respectively) pregnancies. SP cell (more ...)
3.6. Increase of apoptosis in the border of SP layer in HO+/− placentas
TUNEL staining was performed to investigate if the reduction of the Het SP cell layer was due to apoptosis. In E14.5 h/Het placentas (n=5–6), fluorescent-labeled apoptotic cells were found primarily in the JZ near the border of the SP layer (Fig. 3B), with a few cells located in the decidua basalis (DB). No apoptotic cells were found in the labyrinth regions. All in all, the number of apoptotic cells in h/Het placentas was higher than that of w/WT placentas (Figs. 3A and 3B).
Fig. 3
Fig. 3
Increase of apoptosis in the SP layer in HO-1+/− placentas. Representative of microscopic images are shown of placentas at E14.5. Placental regions are outlined by dashed line(s) and indicated as SP cells, GC (glycogen cells), and DB regions. (more ...)
Prussian blue iron staining was performed to see if there was heme deposition or heme-induced toxicity in the Het placentas due to HO-1 deficiency. A few (<3) bright blue staining regions were observed in the JZ, but no difference between w/WT and h/Het placentas were observed (Figs. 3C and 3D), suggesting that no significant heme deposition or toxicity in the Het placentas.
Using DBA staining, we found that brown-stained uNK cells were granulated and distributed in DB and myometrial regions in E14.5 placentas. Distribution of uNK cells were similar between w/WT and h/Het placentas without infiltration into the JZ or labyrinth (Figs. 3E and 3F), suggesting that uNK does not play a role in SP cell layer reduction.
Spiral arteries were visualized in proximal DB region of serially sectioned placentas following H&E staining. Diameters of spiral arteries in Het placentas (Fig. 3H) were relatively smaller than those in WT placentas (Fig. 3G).
3.7. Association of HO-1 deficiency and on the expression of other placental factors
To further elucidate if HO-1 deficiency is coupled with changes in other placental factors, expression levels in iNOS and eNOS (NO synthase system), P21 (cyclin-dependent kinase inhibitor), Mash-2 (transcriptional factor critical for SP formation), neuropilin-1 (marker for T regulatory cells), and Bag-1 (anti-apoptotic/cytoprotective molecule) were quantitated by qRT-PCR or by Western blot. At E15.5, no significant differences between w/WT and h/WT placentas were found for any of the placental factors examined. However, compared to those of w/WT and h/WT, h/Het placentas had higher mRNA levels of iNOS (39% and 49%, respectively), eNOS (14% and 11%, respectively) (Fig. 4A), and Mash-2 (60% and 61%, respectively) (Fig. 4B). No significant changes in levels of P21 and neuropilin-1 mRNA and of Bag-1 protein were detected (data not shown).
Fig. 4
Fig. 4
Effect of HO-1 deficiency on other placental factors. Placentas from w/WT (black, n=3), h/WT (dark grey, n=4), and h/Het (light grey, n=4) embryos were collected at E15.5. (A) Upregulation of iNOS and eNOS expression in placentas from h/Het embryos compared (more ...)
3.8. Changes in blood pressure during HO+/− pregnancies
In order to study if HO-1 deficiency results in gestational hypertension, maternal blood pressures during WT and Het pregnancies were measured from E12.5–E18.5. No difference in systolic blood pressure was found between Het and WT pregnancies; however, a significant increase in diastolic blood pressure was detected in Het pregnancies compared to WT pregnancies (Fig. 5A). Also, no significant difference in blood pressure was found between non-pregnant WT and Het mice (data not shown).
Fig. 5
Fig. 5
Maternal diastolic blood pressures and sFlt-1 levels in WT:W T and Het:Het pregnancies. (A) Diastolic blood pressures in pregnant WT (n=6, solid line) or Het (n=8, dash line) mice from E12.5–E18.5. (B) Maternal plasma sFlt1 levels normalized by (more ...)
3.9. Increase in maternal sFlt-1 level during HO+/− pregnancies
In both WT and Het pregnancies, maternal sFlt-1 levels increased gradually, peaking at E17.5±1, and returning to non-pregnant levels after delivery. Because litter numbers for WT (n~9) and Het (n~5.1) pregnancies were different and since sFlt1 is secreted primarily from the placenta, we normalized sFlt-1 levels to embryo number and found a larger increase in sFlt-1/embryo in the Het pregnancies, suggesting that Het placentas produce more sFlt-1 than WT placentas.
Antioxidative, anti-inflammatory and anti-apoptotic effects mediated by HO-1 have been reported in many tissues [6,30]. In this study, we showed that a complete deficiency of HO-1 expression is associated with early embryonic death, suggesting a critical role of HO-1 in early placentation or embryonic development. Compared to the 5% survival rate of HO-1−/− embryos reported by Poss et al [17] in BALB/c mice, we found an even lower survival rate (2.4%) in FVB mice, indicating that the severity of HO-1 deficiency is strain-dependent. Another cause of fetal resorptions may be due complement activation, which may be affected by a deficiency in HO-1, and deserves further study. Because of this fetal lethality, we focused on the HO+/− genotype to investigate the role of HO-1 on placental development during mid to late gestational ages.
The partial HO-1 deficiency resulted in delayed placental and embryonic development (Table 1). In Het breedings, placental and embryo weights varied more than those of WT breedings, which may be due to an increase in intrauterine abortions and uneven litter numbers on each uterus. An embryo’s in utero location and position by an aborted fetus may have also affected placental and its development. Even with these variable influences, we still observed significant differences in the placentas among w/WT, h/WT, and h/Het embryos at E12.5 and E15.5. Interestingly, the discrepancies were not as pronounced at late pregnancy stages (E18.5) or after birth. In addition, we observed a parallel profile in HO-1 expression (Fig. 1) and placental histology (Fig. 2), in which the defects were significant at E12.5 and E15.5, but not at E18.5, suggesting that the most vulnerable period in placental development occurs at mid-gestational ages when HO-1 expression is high. This vulnerability may be exacerbated as the degree of HO-1 deficiency is enhanced, such as in Het/WT and Het/Het embryos. Because the placenta is an organ comprised of both maternal and fetal tissues, comparisons of placentas arising from WT:Het breedings versus those from Het:Het breedings, may reveal if the deficiency in HO-1 of the Het/Mut embryo contributes more to the development of pregnancy disorders than the deficiency of HO-1 in the mother.
The observed growth delay of the placentas and embryos at E18.5 was diminished, implying that the state of chronic HO-1 deficiency may be substantially compensated by an upregulation of other factors, such as HO-2 (Figs. 1B and 1E), iNOS, eNOS and/or Mash-2 (Figs. 4A and 4B). By qRT-PCR, we found that both HO-1 and HO-2 are highly expressed in WT placentas with levels were very comparable, i.e. neither of them dominated the expression (data not shown). Except for the heme-binding domain, HO-2 shares little similarity with HO-1, such as in primary structure, gene organization, or transcriptional regulation [24,31]. HO-2 has been recognized as the constitutively-expressed HO isozyme in most of the tissues and is only induced by very few stimuli, such as adrenal glucocorticoid [32,33]. However, we did observe a reduction in HO-2 expression when HO-1 was highly induced by a potent HO-1 inducer (cadmium chloride) in cell culture studies (unpublished data) suggesting that there is a balance or equilibrium between HO-1 and HO-2 expression levels. HO-2 is also an oxygen sensor for a calcium-sensitive potassium channel [34] and regulates channel activity during oxygen deprivation. The exact role of HO-2 in oxygen sensing or CO signaling in placental development is yet to be elucidated.
Besides HO-2, both iNOS and eNOS were induced in the Het placentas. NOS/NO system shares many similarities with HO/CO system and their interrelationship has attracted much attention. iNOS, like HO-1, is inducible in many cell types; while eNOS, like HO-2, is relatively constitutively expressed mostly in endothelial cells [35]. Both NO and CO react with the prosthetic heme group of soluble guanylyl cyclase (sGC) and enhance production of intracellular molecular cGMP, to regulate vascular tone [36]. NO can also induce the suppression of apoptosis and inflammation in hepatocytes and macrophages by inducing HO-1 and hence CO production [37]. Expression of eNOS and iNOS was found significantly reduced in the trophoblastic cells of placentas of infants with IUGR [38], suggesting important roles in the pathogenesis of pregnancy disorders. The biochemical mechanisms and pathophysiological significance of HO/CO and NOS/NO pathways in placental development should be further studied.
HO-1 was expressed primarily in the SP regions of WT placentas [18] (Fig. 3E). Notably, the most pronounced defects of HO-1 deficiency were also found in the same regions (Fig. 2). Our observations from TUNEL assays and Prussian blue iron and DBA stainings suggests that the reduction of the SP layer in the h/Het placenta may be associated with an increase in apoptosis (Figs. 3A and 3B), which appears to be not due to heme accumulation/heme toxicity or uNK cell infiltration (Figs. 3C to 3F). However, in the only reported case of human HO-1 deficiency, Koizumi et al [39] showed that the increased heme level and tissue heme deposition in this patient was directly due to the lack of HO-1 enzyme. Since our mouse model (HO-1 Het pregnancy) has partial deficiency in HO-1, the tissue heme accumulation and toxicity were not as severe. Although the precise function of the SP layer is not well understood, the maintenance of its integrity is vital for fetal viability [40]. It could act as a structural support for the developing villous structure of the labyrinth. In addition, since it is traversed by a central maternal artery and lateral maternal veins, it may serve to provide maternal blood to the labyrinth and nutrients to the fetal circulatory system. In addition, it may be a mediator in the exchange and processing information between the maternal and fetal sides. Because Mash-2 is one of the very few genes that have been identified to play a critical role for SP layer formation, its deficiency may cause a lack of a SP layer and subsequent fetal death [41]. Our observed upregulation in Mash-2 mRNA levels in Het placentas may by a compensatory response in HO-1 deficiency, although the difference did not reach statistical significance.
Previous work by Zenclussen et al. has shown that an overexpression of HO-1 induced by cobalt protoporphyrin (CoPP) could prevent fetal rejection in an allopregnancy mouse model through the upregulation of neuropilin, a marker for T regulatory cells, and Bag-1, an anti-apoptotic/cytoprotective molecule [21]. Acute suppression of HO activity by an HO inhibitor, zinc protoporphyrin (ZnPP), did not change the expression of neuropilin-1, but decreased Bag-1 protein and mRNA levels. In this study, we showed that levels of neuropilin or Bag-1 did not significant change in HO-1+/− placentas. These discrepant findings may be due to the different mouse models used as well as to conditions of an acute (a single exposure to a drug) vs. a chronic HO-1 deficiency (genetic). Acute inhibition by HO chemical inhibitors suppress not only HO-1, but also HO-2 activity. Moreover, they can also affect the expression of other factors, such as NOS or sGC.
Our data has shown that a deficiency of HO-1 in pregnancy can induce elevations in maternal diastolic blood pressures, suggesting that HO-1 plays a role in maternal vascular tone regulation. Relatively less dilated spiral arteries were also detected in Het placentas (Figs. 3H and 3G). It is still not understood why HO-1 deficiency results in smaller spiral arteries and whether the change is just proportional to the overall smaller size of Het placentas, or directly due to defects that may induce pre-eclampsia-like symptoms. Moreover, a significant increase in plasma sFlt-1 levels of pregnant Het mice were also observed, which is consistent with studies by Cudmore et al [25], who showed that HO-1 negatively regulates sFlt-1 expression in pre-eclampsia. However, we did not observe any systolic hypertension or increases in the urinary protein/creatinine ratio (data not shown), indicating that a partial deficiency in HO-1 is not sufficient to cause the full spectrum of pre-eclampsia-like symptoms. It has been well accepted that the pathogenesis of pre-eclampsia is not due solely to a single mechanism. Because, our Het mice model reflects a state of chronic and partial HO-1 deficiency, resembling that of the human HO-1 deficiency circumstance, studies using this model may advance the understanding of the role of HO in placental development and in the maintenance of pregnancy as well as the possible link of HO-1 deficiency to pregnancy disorders, including pre-eclampsia.
ACKNOWLEDGEMENTS
This work was supported by the Christopher Hess Research Fund, and the Mary L. Johnson Research Fund. We thank Mrs. Pauline Chu, and Drs Yuan Cao and Stacy Burns-Guydish for their technical help. We also thank AC Zenclussen for providing us with neuropilin primers for qRT-PCR.
Footnotes
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Summary sentence: Heme oxygenase-1 plays an important role in the development of the placenta and the maintenance of a healthy pregnancy.
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