Antioxidative, anti-inflammatory and anti-apoptotic effects mediated by HO-1 have been reported in many tissues [6
]. 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 (). 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 () and placental histology (), 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 (), iNOS, eNOS and/or Mash-2 (). 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 [2
]. 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
]. 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
] (). Notably, the most pronounced defects of HO-1 deficiency were also found in the same regions (). 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 (), which appears to be not due to heme accumulation/heme toxicity or uNK cell infiltration (). 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 (). 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.