Haem oxygenase-1 dictates intrauterine fetal survival in mice via carbon monoxide.

Pregnancy establishment implies the existence of a highly vascularized and transient organ, the placenta, which ensures oxygen supply to the fetus via haemoproteins. Haem metabolism, including its catabolism by haem oxygenase-1 (HO-1), should be of importance in maintaining the homeostasis of haemoproteins and controlling the deleterious effects associated with haem release from maternal or fetal haemoglobins, thus ensuring placental function and fetal development. We demonstrate that HO-1 expression is essential to promote placental function and fetal development, thus determining the success of pregnancy. Hmox1 deletion in mice has pathological consequences for pregnancy, namely suboptimal placentation followed by intrauterine fetal growth restriction (IUGR) and fetal lethality. These pathological effects can be mimicked by administration of exogenous haem in wild-type mice. Fetal and maternal HO-1 is required to prevent post-implantation fetal loss through a mechanism that acts independently of maternal adaptive immunity and hormones. The protective HO-1 effects on placentation and fetal growth can be mimicked by the exogenous administration of carbon monoxide (CO), a product of haem catabolism by HO-1 that restores placentation and fetal growth. In a clinical relevant model of IUGR, CO reduces the levels of free haem in circulation and prevents fetal death. We unravel a novel physiological role for HO-1/CO in sustaining pregnancy which aids in understanding the biology of pregnancy and reveals a promising therapeutic application in the treatment of pregnancy pathologies.

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.

Mechanisms behind flare of renal lupus during murine pregnancy.

The outcome of pregnancy in systemic lupus erythematosus is still controversial. The authors recently reported the disappearance of the manifestation of the skin disease but a diminished survival rate in lupus-prone animals undergoing several pregnancies. It was postulated that lupus-prone animals must have subclinical renal symptoms at an early age and that immune and hormonal changes during pregnancy exacerbate immune reactions in the kidneys, leading to a shortened life span. Here, the authors analysed changes at day 14 of pregnancy in lupus-prone LPR (MRL/lpr) mice and MRL controls regarding cytokines, regulatory T (Treg) cells and deposition of immunocomplexes. Worsened kidney function was observed during pregnancy, even in the absence of lupus signs. This was accompanied by renal inflammation and higher interferon-gamma and interleukin-10 levels. C3 and immunoglobulin G deposition was enhanced in kidney and placenta from lupus-prone pregnant animals. Pregnancy enhanced the levels of Treg cells in control animals but not in lupus-prone animals. As pregnancy-induced Treg cells were shown to be specific for paternal antigens it is not to be expected that these Treg cells can help to destroy autoreactive cells. The authors conclude that early subclinical kidney disease in lupus-prone animals exacerbates during pregnancy. Albeit obtained with an experimental animal model, their data are potentially of importance for lupus patients of reproductive age.

Tacrolimus depresses local immune cell infiltration but fails to reduce cortical contusion volume in brain-injured rats.

The immunosuppressant drug tacrolimus (FK-506) failed to show an anti-edematous effect despite suppressing pro-inflammatory cytokines in cerebrospinal fluid following focal traumatic brain injury. By questioning the role of the inflammatory response as a pharmacological target, we investigated the effects of FK-506 on immune cell infiltration in brain-injured rats. Following induction of a cortical contusion, male Sprague-Dawley rats received FK-506 or physiological saline intraperitoneally. Brains were removed at 24 h, 72 h or 7 days, respectively. Frozen brain sections (7 microm) were stained immunohistologically for markers of endothelial activation (intercellular adhesion molecule-1--ICAM-1), neutrophil infiltration (His-48), and microglial and macrophage activation (Ox-6; ED-1), respectively. Immunopositive cells were counted microscopically. Contusion volume (CV) was quantified morphometrically 7 days after trauma. Inflammatory response was confined to the ipsilateral cortex and hippocampal formation, predominating in the contusion and pericontusional cortex. Strongest ICAM-1 expression coincided with sustained granulocyte accumulation at 72h which was suppressed by FK-506. Ox-6+ cells prevailing at 72 h were also significantly reduced by FK-506. ED-1+ cells reaching highest intensity at 7 days were significantly attenuated at 72 h. Cortical CV was not influenced. FK-506 significantly decreased post-traumatic local inflammation which, however, was not associated with a reduction in cortical CV. These results question the importance of post-traumatic local immune cell infiltration in the secondary growth of a cortical contusion.

Ubiquitin reduces contusion volume after controlled cortical impact injury in rats.

Recent data suggest that ubiquitin has anti-inflammatory properties and therapeutic potential after severe trauma and brain injuries. However, direct evidence for its neuroprotective effects has not yet been provided. We hypothesized that ubiquitin treatment is neuroprotective, and thus reduces brain edema formation and cortical contusion volume after closed traumatic brain injuries. To test this hypothesis, a focal cortical contusion was induced using a controlled cortical impact (CCI) model in Sprague-Dawley rats. Animals (n = 27) were randomized to either 1.5 mg/kg ubiquitin or vehicle (placebo) intravenously within 5 min after CCI. Blood pressure, arterial blood gases (ABG) and intracranial pressure (ICP) were monitored. Ubiquitin serum and cerebrospinal fluid levels were measured by ELISA. Brain water content was quantified gravimetrically after 24 h and cerebral contusion volume was determined in triphenyltetrazolium-chloride stained brains after 7 days. All animals recovered to normal activity. ICP and cerebral perfusion pressures were normal at the end of the observation period. Ubiquitin serum and CSF levels at 24 h and 7 days after CCI were similar in both groups. With ubiquitin brain water content of the injured hemisphere was slightly lower (n = 6/group; 79.97 +/- 0.29% vs. 81.11 +/- 0.52%; p = 0.08). Cortical contusion volume was significantly lower with ubiquitin (n = 7-8/group; 32.88 +/- 2.1 mm(3) vs. 43.96 +/- 4.56 mm(3); p = 0.025). This study shows that ubiquitin treatment after brain injury has direct neuroprotective effects, as demonstrated by improved brain morphology 7 days after brain injury. In connection with its beneficial effects in our previous studies, these data suggest ubiquitin as a promising candidate protein therapeutic for the treatment of brain injuries.

Effects of pigment epithelium-derived factor on traumatic brain injury.

PURPOSE: Pigment epithelium-derived factor (PEDF) is a multifunctional protein with antiangiogenic, anti-inflammatory, neurotrophic and neurogenic properties. The effect of PEDF on traumatic brain injury (TBI) has not been explored. In this study, we aimed to show the in vivo effects of PEDF on lesion volume, cell death and cell proliferation after TBI. METHODS: Rats were subjected to controlled cortical impact injury (CCII). PEDF mRNA brain levels were measured by RT-PCR. The lesion volume, cell proliferation, cell death and microglia activation were assessed in the brains of lesioned animals with intraventricular alzet infusion of PEDF or aCSF, and intraperitoneal injections of BrdU. RESULTS: We detected a significant increase of PEDF mRNA levels after TBI. PEDF intraventricular infusion showed no significant effect on the contusion volume, whereas the number of dead cells, activated microglia, BrdU-positive cells around the lesion were significantly decreased. In contrast, PEDF application increased cell proliferation in the ipsilateral subventricular zone. No effect was found on cell proliferation in the dentate gyrus. CONCLUSION: The present work indicates that PEDF acts as a multifunctional agent after TBI influencing cell death, inflammation and cell proliferation.

Hormonal Fluctuations during the Estrous Cycle Modulate Heme Oxygenase-1 Expression in the Uterus.

Deletion of the heme oxygenase-1 (HO-1) (Hmox1) locus in mice results in intrauterine lethality. The expression of the heme catabolizing enzyme encoded by this gene, namely HO-1, is required to successfully support reproductive events. We have previously observed that HO-1 acts at several key events in reproduction ensuring pregnancy. HO-1 defines ovulation, positively influences implantation and placentation, and ensures fetal growth and survival. Here, we embarked on a study aimed to determine whether hormonal changes during the estrous cycle in the mouse define HO-1 expression that may influence receptivity. We analyzed the serum levels of progesterone and estrogen by ELISA and HO-1 mRNA expression in uterus by real time RT-PCR at the metestrus, proestrus, estrus, and diestrus phases of the estrous cycle. Further, we studied the HO-1 protein expression by western blot upon hormone addition to cultured uterine AN3 cells. We observed that HO-1 variations in uterine tissue correlated to changes in hormonal levels at different phases of the estrus cycle. In vitro, HO-1 protein levels in AN3 cells augmented after the addition of physiological concentrations of progesterone and estradiol, which confirmed our in vivo observations. Our data suggest an important role for hormones in HO-1 regulation in uterus during receptivity, a process known to have a significant impact in receptivity and later on blastocyst implantation.

Regeneration of nucleus pulposus tissue in an ovine intervertebral disc degeneration model by cell-free resorbable polymer scaffolds.

Degeneration of intervertebral discs (IVDs) occurs frequently and is often associated with lower back pain. Recent treatment options are limited and treat the symptoms rather than regenerate the degenerated disc. Cell-free, freeze-dried resorbable polyglycolic acid (PGA)-hyaluronan implants were used in an ovine IVD degeneration model. The nucleus pulposus of the IVD was partially removed, endoscopically. PGA-hyaluronan implants were immersed in autologous sheep serum and implanted into the disc defect. Animals with nucleotomy only served as controls. The T2-weighted/fat suppression sequence signal intensity index of the operated discs, as assessed by magnetic resonance imaging (MRI), showed that implantation of the PGA-hyaluronan implant improved (p = 0.0066) the MRI signal compared to controls at 6 months after surgery. Histological analysis by haematoxylin and eosin and safranin O staining showed the ingrowth of cells with typical chondrocytic morphology, even cell distribution, and extracellular matrix rich in proteoglycan. Histomorphometric analyses confirmed that the implantation of the PGA-hyaluronan scaffolds improved (p = 0.027) the formation of regenerated tissue after nucleotomy. Disc heights remained stable in discs with nucleotomy only as well as after implantation of the implant. In conclusion, implantation of cell-free polymer-based implants after nucleotomy induces nucleus pulposus tissue regeneration and improves disc water content in the ovine model.

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.

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.

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.

Blockage of heme oxygenase-1 abrogates the protective effect of regulatory T cells on murine pregnancy and promotes the maturation of dendritic cells.

Regulatory T cells (Treg) play an important role in fetal protection. They expand during normal pregnancy and protect fetal antigens from maternal effector cells. Their effect is associated with the up-regulation of tolerance-associated molecules at the fetal-maternal interface. Among these, Heme Oxygenase-1 (HO-1, coded by Hmox1) is of special importance as its blockage correlates with increased abortion rates and its up-regulation positively affects pregnancy outcome. Here, we aimed to investigate whether the protective effect of Treg is mediated by HO-1 in a mouse model. HO-1 blockage by Zinc Protoporhyrin (ZnPPIX) abrogated the protective effect of Treg transfer. We found that HO-1 is important in maintaining maternal dendritic cells (DCs) in an immature state, which contributes to the expansion of the peripheral Treg population. This brings to light one essential pathway through which Treg mediates the semi-allogeneic fetus tolerance.

Pregnancy: tolerance and suppression of immune responses.

Presence of foreign tissue in a host's body would immediately lead to a strong immune response directed to destroy the alloantigens present in fetus and placenta. However, during pregnancy, the semiallogeneic fetus is allowed to grow within the maternal uterus due to multiple mechanisms of immune tolerance, which are discussed in this chapter.

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.

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.

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.

Anti-P- and E-selectin therapy prevents abortion in the CBA/J x DBA/2J combination by blocking the migration of Th1 lymphocytes into the foetal-maternal interface.

Leukocyte migration into inflamed tissues comprises dynamic interactions between immune and endothelial cells through events controlled by adhesion molecules, e.g., P- and E-selectins, which mediate Th1 cells recruitment after injury. Since miscarriage is known to be a Th1 event and selectins are expressed at the murine foetal-maternal interface, the purpose of our study was to investigate whether blocking P- and E-selectins before implantation could inhibit Th1 migration into the foetal-maternal interface and thus prevent foetal rejection. DBA/2J-mated CBA/J females were treated with monoclonal antibodies (mAbs) against P-selectin or with both, anti-P- and anti-E-selectins combined on days 2 and 4 of pregnancy. PBS-treated females served as controls. Our data revealed a significant improvement in pregnancy outcome in both treated groups compared to the control, which is due to the effectiveness of the mAb against P-selectin, since the treatment with anti-E-selectin alone could not prevent abortion. We further observed that there was diminished Th1 cytokine production by decidual immune cells in all treated groups in comparison to the controls. Our data first confirm the important role of P-selectin in mediating the extravasation of abortive cells, while opening new therapeutic opportunities.

Pre-eclampsia is not associated with changes in the levels of regulatory T cells in peripheral blood.

PROBLEM: The acceptance of the semi-allogeneic fetus within the maternal environment requires tolerance mechanisms not fully characterized yet. Normal pregnancy is known to be associated with a Th2 profile. Furthermore, regulatory T cells (Tregs) were proposed to regulate the Th2/Th1 balance at early stages of pregnancy. Treg may avoid the shift to a Th1 profile, thus preventing miscarriage. Accordingly, spontaneous abortion is characterized by a Th1 dominance and diminished levels of Treg. The major aim of the present work was to investigate if pre-eclampsia, a late immunological complication of pregnancy, is characterized by similar hallmarks. METHOD OF STUDY: We measured the surface antigens CD4, CD25, CD8 and CTLA4 in peripheral blood from patients suffering from pre-eclampsia (n = 8) and age-matched patients undergoing normal pregnancies (n = 9) by four-color flow cytometry. RESULTS: We were not able to find any significant differences in the levels of CD4(+), CD25(+), CD8(+), CTLA4, CD4(+)/CD25(+), CD4(+)/CD25(bright), CD4(+)/CTLA4, CD25(+)/CTLA4, CD4(+)/CD25(+)/CTLA4, CD8(+)/CD25(+), CD8(+)/CTLA4 or CD8(+)/CD25(+)/CTLA4 cell subsets. CONCLUSIONS: Our data confirm comparable number of Tregs during pre-eclampsia and normal pregnancy in peripheral blood. Other regulatory mechanisms might be involved during late pregnancy.