Heme oxygenase-1 is critically involved in placentation, spiral artery remodeling, and blood pressure regulation during murine pregnancy.

The onset of pregnancy implies the appearance of a new organ, the placenta. One main function of the placenta is to supply oxygen to the fetus via hemoproteins. In this review, we highlight the importance of the enzyme heme oxygenase-1 (HO-1) for pregnancy to be established and maintained. HO-1 expression is pivotal to promote placental function and fetal development, thus determining the success of pregnancy. The deletion of the gene Hmox1 in mice leads to inadequate remodeling of spiral arteries and suboptimal placentation followed by intrauterine growth restriction (IUGR) and fetal lethality. A partial Hmox1 deletion leads to IUGR as well, with heterozygote and wild-type fetuses being born, but Hmox1 (-/-) significantly below the expected Mendelian rate. This strong phenotype is associated with diminished number of pregnancy-protective uterine natural killer (uNK) cells. Pregnant heterozygote females develop gestational hypertension. The protective HO-1 effects on placentation and fetal growth can be mimicked by the exogenous administration of carbon monoxide (CO), a product of heme catalyzed by HO-1. CO application promotes the in situ proliferation of uNK cells, restores placentation and fetal growth, while normalizing blood pressure. Similarly, HO-1 inhibition provokes hypertension in pregnant rats. The HO-1/CO axis plays a pivotal role in sustaining pregnancy and aids in the understanding of the biology of pregnancy and reveals a promising therapeutic application in the treatment of pregnancy complications.

Exploring the potential of low doses carbon monoxide as therapy in pregnancy complications.

Heme Oxygenase-1 (HO-1) has been shown to play a pivotal role in pregnancy outcome and its ablation leads to abnormal placentation, intrauterine fetal growth restriction (IUGR) and subsequent intrauterine fetal death. Carbon monoxide (CO) has been found to mimic the protective effects of HO-1 activity, rescuing HO-1-deficient fetuses. This gasotransmitter arises in biological systems during the oxidative catabolism of heme by HO. Here, we explored the potential of CO in preventing IUGR and established the optimal doses and therapeutic time window in a clinically relevant mouse model. We additionally investigated the pathways activated upon CO application in vivo. We established 50 ppm as the best lowest dose of CO necessary to prevent growth restriction being the optimal time frame during days 3 to 8 of mouse pregnancy. CO lead to higher fetal and placental weights and avoided fetal death without showing any pathologic effects. CO breathing further suppressed inflammatory responses, diminished placenta apoptosis and complement deposition and regulated placental angiogenesis. Our results confirm the protective role of the HO-1/CO axis and point this gas as an emerging therapeutic possibility which is worth to further explore.

Heme oxygenase-1 expression in the ovary dictates a proper oocyte ovulation, fertilization, and corpora lutea maintenance.

PROBLEM: Animals deficient in Heme oxygenase-1 (HO-1, Hmox1(-/-) mice) have impaired pregnancies, characterized by intrauterine fetal death. HO-1 expression has been shown to be essential for pregnancy by dictating placentation and intrauterine fetal development. Its absence leads to intrauterine fetal growth restriction and fetal loss, which is independent of the immune system. Defect in previous steps, e.g., ovulation, may, however, also count for their poor reproductive outcome. METHOD OF STUDY: Here, we investigated ovulation after hormonal hyperstimulation in Hmox1 wild-type and knockout animals. RESULTS AND CONCLUSIONS: We observed that animals lacking HO-1 produced significantly less oocytes after hormonal stimulation than wild type animals and this was mirrored by the number of corpora lutea in the ovary. Furthermore, ovulated oocytes from Hmox1(-/-) animals were poorly fertilized compared with those from wild-type animals. In conclusion, we demonstrate here that HO-1 plays a pivotal role in the process of oocyte ovulation as well as fertilization, bringing to light a new and unsuspected role for HO-1.

The effect of ubiquitin on immune response after controlled cortical impact injury.

BACKGROUND: Previously, we reported that exogenous ubiquitin reduces cortical contusion volume and tends to reduce brain water content after controlled cortical impact injury Controlled Cortical Impact Injury (CCII) in rats. The mechanisms how exogenous ubiquitin exerts these effects remain unclear. Some studies revealed ubiquitin's immune modulatory abilities; therefore, we hypothesized that ubiquitin influences the local innate inflammatory response after CCII. METHODS: Sprague-Dawley rats were exposed to CCII and randomized to either 1.5 mg/kg ubiquitin or 0.9% NaCl intravenously within 5 minutes after CCII. Immune cells were immunohistochemically stained with OX-42, myeloperoxidase (MPO), HIS48, ED1, and glial fibrillary acidic protein (GFAP). Apoptosis was analyzed by using terminal desoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL). Levels of interleukin (IL)-1ß, IL-6, IL-10, tumor necrosis factor (TNF)-α, and IL-1 receptor antagonist (IL-1ra) were quantified using real-time reverse-transcriptase polymerase chain reaction (rt-PCR). RESULTS: ED1-positive cells were significantly increased in the pericontusional cortex after ubiquitin treatment at day 7 (823±182 cells/mm² vs. 550±246 cells/mm²; p=0.04). IL-10 expression after 3 days was significantly lower in the verum group (1.065¹°â»5±0.6093¹°â»5 vs. 2.266¹°â»5±1.244¹°â»5 relative messenger RNA expression; p=0.04) and TNF-α-levels tended to be higher in the verum group (22.01¹°â»5±10.87¹°â»5 vs. 9.34¹°â»5±4.44¹°â»5 relative messenger RNA; p=0.096). Quantification of apoptotic cells did not differ between the groups. CONCLUSION: Exogenous ubiquitin modulates the immune response by influencing the infiltration of macrophages or activated microglia and the expression of IL-10 and possibly TNF-α after CCII. The effects of these changes in immune response on posttraumatic neurodegeneration still need to be clarified.

Intervertebral disc regeneration after implantation of a cell-free bioresorbable implant in a rabbit disc degeneration model.

Degeneration of the intervertebral disc is the most common cause of lower back pain. Interestingly, all available treatments are limited to treat the symptoms and not the underlying biologic alterations of the disc. Freeze-dried resorbable non-woven polyglycolic acid (PGA) - hyaluronan implants were used in a degenerated disc disease (DDD) model in New Zealand white rabbits. The constructs were immersed in allogenic serum and implanted into the disc defect. Animals with discectomy only served as controls. The T2-weighted/fat suppression sequence signal intensity of the operated discs as assessed by magnet resonance imaging decreased in both groups one week after the operation compared to a healthy disc. After 12 months the implanted group showed an increase of 51% in the signal intensity compared to the 1-week results whereas the signal intensity in the sham group remained on the same level from one week to 12 months. Histological and quantitative immunohistochemical examination after 12 months indicated cell migration into the defect and showed formation of disc repair tissue. In controls, repair tissue containing type II collagen was not evident. In conclusion, the implantation of polymer-based constructs after discectomy induces tissue regeneration resulting in improvement of the disc water content.

The persistence of paternal antigens in the maternal body is involved in regulatory T-cell expansion and fetal-maternal tolerance in murine pregnancy.

PROBLEM: Mammalian pregnancy is a state of immunological tolerance and CD4(+) CD25(+) regulatory T cells (Treg) contribute to its maintenance. Knowing that Treg act in an antigen-specific way during pregnancy, we hypothesized that they are generated after maternal immune cells encounter paternal antigens. METHOD OF STUDY: We mated wild type females with transgenic green fluorescent protein (GFP) males in an allogenic setting and killed them on different days of pregnancy. RESULTS: Presence of paternal and maternal MHC class II(+) cells in vaginal lavage on day 0.5 of pregnancy was confirmed. Thus, antigen presentation may take place early during pregnancy in the periphery either by the direct or indirect pathways. Foxp3(+) cells known to have regulatory activity could be detected on day 2 of pregnancy in lymph nodes and shortly after implantation at the fetal-maternal interface. CONCLUSION: Our data suggest that paternal antigens are processed early during pregnancy, which leads to the generation of Treg. The continuous release of placental antigens into the maternal circulation allows the maintenance of a Treg population which is specific for paternal antigens and mediates tolerance toward the semi-allogeneic fetus until the time point of birth.

Supporting the hypothesis of pregnancy as a tumor: survivin is upregulated in normal pregnant mice and participates in human trophoblast proliferation.

PROBLEM: Survivin, a tumor-promoting antiapoptotic molecule, is expressed in the human placenta. Here, we analyzed its expression during normal and pathological murine pregnancy and investigated its participation in human first trimester trophoblast cell survival and proliferation. METHOD OF STUDY: We first analyzed the expression of survivin on the mRNA and protein level at the fetal-maternal interface of normal pregnant (CBA/J x BALB/c) and abortion-prone (CBA/J x DBA/2J) mice at different pregnancy stages by RT-PCR and immunohistochemistry. We also evaluated apoptosis in murine trophoblasts in both mating combinations by TUNEL technique. Functional studies were carried out by knockdown survivin by means of siRNA methodology in two human first trimester trophoblast cell lines [Swan.71 (Sw.71) and HTR8 (H8)]. RESULTS: We observed a peak in mRNA levels on day 5 and a peak of protein levels on day 8 of pregnancy in both combinations. The level of survivin in animals from the abortion-prone group was decreased compared with normal pregnant mice on day 8, which was accompanied by elevated apoptosis rates. In later pregnancy stages (days 10 and 14), survivin levels decreased to levels comparable to those observed right after fecundation in both groups. Transfection of human first trimester cell lines (H8 and Sw.71) with siRNA targeting the survivin gene led to a 76-82% reduction of its expression leading to reduced trophoblast cell viability and proliferation. CONCLUSION: Our findings suggest an important role of survivin to promote trophoblast cell survival and proliferation during placentation, thus maintaining pregnancy. The pregnancy-associated expression of a cancer molecule such as survivin supports the 'pseudo-malignancy' hypothesis of pregnancy. Our data may contribute to the better understanding of trophoblast cell development during implantation and placentation.