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Heme oxygenase-1 (HO-1) is an evolutionarily conserved stress response enzyme and important in pregnancy maintenance, fetal and neonatal outcomes, and a variety of pathologic conditions. Here, we investigated the effects of an exposure to systemic inflammation late in gestation [embryonic day (E)15.5] on wild-type (Wt) and HO-1 heterozygous (Het, HO-1+/-) mothers, fetuses, and offspring. We show that alterations in fetal liver and spleen HO homeostasis during inflammation late in gestation can lead to a sustained dysregulation of innate immune cell populations and intracellular myeloid HO-1 expression in the spleen through young adolescence [postnatal day 25] in mice., Competing Interests: The authors have declared no competing interests exist.

Introduction: Severe hypoxia exists in placentas during early pregnancy, with reoxygenation during mid-gestation. Hypoxia-inducible factor-1α (Hif1α), an oxygen sensor, initiates placental vascular development. We have shown that the placental vasculature in Hmox1-deficient (Hmox1 +/- , Het) pregnancies is impaired, with morphological defects similar to Hif1α-deficient placentas., Materials and Methods: Whole wild-type (WT) and Het mouse placentas were collected at E8.5 (1%-3% O 2 ) and E9.5-15.5 (8%-10% O 2 ). mRNA levels were determined using real-time RT-PCR or PCR arrays and protein levels using Western blot. Bone marrow-derived macrophages (BMDMs) from WT, Het, and Hmox1 knockout (KO) mice, representing different Hmox1 cellular levels, were generated to study the role of Hmox1 on Hif1α 's response to hypoxia-reoxygenation and gestational age-specific placental lysates., Results: Hif1α was expressed in WT and Het placentas throughout gestation, with protein levels peaking at E8.5 and mRNA levels significantly upregulated from E9.5-E13.5, but significantly lower in Het placentas. Genes associated with angiogenesis (Vegfa, Vegfr1, Mmp2, Cxcl12, Angpt1, Nos3), antioxidants (Sod1, Gpx1), and transcription factors (Ap2, Bach1, Nrf2) were significantly different in Het placentas. In response to in vitro hypoxia-reoxygenation and to WT or Het placental lysates, Hif1α transcription was lower in Het and Hmox1 KO BMDMs compared with WT BMDMs., Discussion: These findings suggest that deficiencies in Hmox1 underlie the insufficient placental Hif1α response to hypoxia-reoxygenation during gestation and subsequently impair downstream placental vascular formation. Therefore, a dysregulation of Hif1α expression caused by any genetic defect or environmental influence in early pregnancy could be the root cause of pregnancy disorders., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: Statins induce heme oxygenase-1 (HO-1) expression in vitro and in vivo. Low HO-1 expression is associated with pregnancy complications, e.g. preeclampsia and recurrent miscarriages. Here, we investigated the effects of pravastatin on HO-1 expression, placental development, and fetal survival in mice with a partial HO-1 deficiency., Methods: At E14.5, untreated pregnant wild-type (WT, n=13-18), untreated HO-1 +/- (Het, n=6-9), and Het mice treated with pravastatin (Het+Pravastatin, n=12-14) were sacrificed. Numbers of viable fetuses/resorbed concepti were recorded. Maternal livers and placentas were harvested for HO activity. Hematoxylin and eosin (H&E) and CD31 immunohistochemical staining were performed on whole placentas., Results: Compared with WT, HO activity in Het livers (65±18%, P<0.001) and placentas (74±7%, P<0.001) were significantly decreased. Number of viable fetuses per dam was significantly lower in Untreated Het dams (6.0±2.2) compared with WT (9.1±1.4, P<0.01), accompanied by a higher relative risk (RR) for concepti resorption (17.1, 95% CI 4.0-73.2). In Hets treated with pravastatin, maternal liver and placental HO activity increased, approaching levels of WT controls (to 83±7% and 87±14%, respectively). The number of viable fetuses per dam increased to 7.7±2.5 with a decreased RR for concepti resorption (2.7, 95% CI 1.2-5.9). In some surviving Untreated Het placentas, there were focal losses of cellular architecture and changes suggestive of reduced blood flow in the labyrinth. These findings were absent in Het+Pravastatin placentas., Discussion: Pravastatin induces maternal liver and placental HO activity, may affect placental function and improve fetal survival in the context of a partial deficiency of HO-1., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Increased bilirubin production due to hemolysis can lead to severe neonatal hyperbilirubinemia and, if left untreated, to bilirubin neurotoxicity. Post-cardiac surgery newborns have been shown to be at an increased risk for developing hyperbilirubinemia and also hemolysis. Isoflurane (ISO), a volatile anesthetic agent routinely used in newborn surgery, has been reported to upregulate heme oxygenase 1 (HO-1) expression. HO is the rate-limiting enzyme in the bilirubin production pathway., Objective: Here, we evaluated whether ISO exposure induces HO-1 and further increases bilirubin production in a hemolytic newborn mouse model., Methods: Three-day-old newborn mice were exposed to 2% ISO for 18 min or air. Liver HO activity and HO-1 protein were measured after exposure to ISO. Next, we evaluated the effect of ISO exposure on bilirubin production as indexed by the total body excretion rate of carbon monoxide following heme loading., Results: ISO significantly increased liver HO activity 120% and 116% at 24 and 48 h, respectively, after exposure. HO-1 protein levels also similarly increased after ISO exposure, but the increases were not statistically significant compared with controls. After heme loading, ISO-exposed pups had significantly higher bilirubin production rates (1.24-fold), and also peaked earlier, than age-matched nonexposed pups., Conclusions: ISO exposure can induce HO-1 expression in the liver and may explain the development of severe hyperbilirubinemia in postsurgical infants, especially in those undergoing hemolysis., (© 2018 S. Karger AG, Basel.)

Background: Sepsis in preterm infants is associated with systemic inflammatory responses. The stress-response protein heme oxygenase-1 (HO-1) has protective anti-inflammatory properties. Recently, we reported a protective role of HO-1 using our non-surgical cecal slurry (CS) model in wild-type (WT) mouse pups. Here, we extend these findings to investigate the association of HO-1 deficiency with sepsis severity., Methods: Adapting the Wynn model, we induced sepsis in 4-day-old HO-1-deficient (HO-1 +/- , Het) pups to determine if HO-1 deficiency affected survival rates at the LD40 (2.0 mg/g) of WT pups. To see if HO-1 induction affected sepsis severity, we gave 30-μmol heme/kg subcutaneously to 3-day-old mice 24 h prior to sepsis induction., Results: Post-sepsis induction, Het pups had a mortality of 85.0% (n = 20) and increased expression of the pro-inflammatory gene in the livers and affected hematologic profiles. Heme treatment 24 h prior to sepsis induction significantly increased liver HO activity, reduced mortality to 24.5% (n = 17), attenuated inflammatory responses, reduced spleen bacterial counts, and significantly increased peripheral neutrophils., Conclusions: A partial deficiency in HO-1 increased the progression and mortality in sepsis. Furthermore, induction of HO-1 significantly reduced the mortality even in Het pups. Thus, we conclude that HO-1 plays an important role in the protection against preterm sepsis.

Problem: Infection during pregnancy can disrupt regulatory/effector immune system balance, resulting in adverse pregnancy and fetal-neonatal outcomes. Heme oxygenase-1 (HO-1) is a major regulatory enzyme in the immune system. We observed maternal immune response dysregulation during late gestational inflammation (LGI), which may be mediated by HO-1. Here, we extend these studies to examine the immune response of offspring., Method of Study: Pregnant wild-type (Wt) and HO-1 heterozygote (Het) dams were treated with lipopolysaccharide (LPS) or vehicle at E15.5. Pups' splenic immune cells were characterized using flow cytometry., Results: CD3 + CD4 + CD25 + (Tregs) and CD3 + CD8 + (Teffs) T cells in Wt and Het were similar in control neonates and increased with age. We showed not only age- but also genotype-specific and long-lasting T-cell dysregulation in pups after maternal LGI. The persistent immune dysregulation, mediated by HO-1 deficiency, was reflected as a decrease in Treg FoxP3 and CD3 + CD8 + T cells, and an increase in CD4 + /CD8 + T-cell and Treg/Teff ratios in Hets compared with Wt juvenile mice after maternal exposure to LGI., Conclusion: Maternal exposure to LGI can result in dysregulation of splenic T cells in offspring, especially in those with HO-1 deficiency. We speculate that these immune alterations are the basis of adverse outcomes in neonates from mothers exposed to low-grade (subclinical) infections., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Preterm sepsis is characterized by systemic bacterial invasion and inflammatory response. Its pathogenesis is unclear due to lack of proper animal models. Heme oxygenase-1 (HO-1) can affect physiologic and pathologic conditions through its anti-inflammatory, antioxidative, and anti-apoptotic properties. Since HO-1 is developmentally regulated, it may play a role in the pathogenesis of preterm sepsis. For this study, sepsis was induced using the non-surgical "cecal slurry" (CS) model. CS was given intraperitoneally at various doses to 4-day-old newborn mice to determine dose-dependent effects. The LD40 was then given and changes in bodyweight, bacterial colonization of organs, hematology, serum biochemistry, and immunomodulatory gene expression were determined. We found a dose-dependent mortality with an LD40 of 2.0 mg/g. Significant bacterial colonization and hematological changes (leukocytopenia, thrombocytopenia, and lymphocytopenia) and increased gene expression of pro-inflammatory cytokines, pattern-recognition receptors, and other genes related to immune responses were also observed. Twenty-four hours post-sepsis induction, bodyweight loss was associated with mortality and organ damage. Finally, to elucidate a protective role of HO-1, 30-μmol heme/kg was given subcutaneously 24 h pre-sepsis induction. HO activity in livers and spleens significantly increased 64% and 50% over age-matched controls 24 h post-heme administration. Importantly, heme significantly reduced mortality from 40.9% to 6.3% (P <0.005) and gene expression of pro-inflammatory cytokines (Ccl5, Cxcl10, IL-1b, and Ifng). We conclude that the CS model can be used as a model to study preterm sepsis. Because induction of HO-1 significantly reduced mortality, we speculate that HO-1 may confer protection against sepsis in preterm infants.

Infiltrating myeloid cells in pregnant uteri play critical roles in the establishment of the placenta and maintenance of normal pregnancies. Their recruitment and proliferation are primarily mediated by the interactions of cytokines and chemokines secreted locally with their corresponding receptors. Heme oxygenase-1 (HO-1) has various physiologic properties that contribute to placental vascular development, with deficiencies in HO-1 associated with pregnancy disorders. Here, we investigated the effect of HO-1 on myeloid cell infiltration into pregnant uteri using a partial HO-1-deficient (Het, HO-1 +/- ) mouse model. With the use of flow cytometry, HO-1 was found predominantly expressed in circulating and uterine myeloid cells, specifically neutrophils and monocytes/macrophages. In pregnant Het uteri, the numbers of neutrophils and monocytes/macrophages were significantly reduced compared with pregnant wild-type (WT; HO-1 +/+ ) uteri. With the use of BrdU in vivo assays, HO-1 deficiency did not affect cell proliferation or blood cell populations. With the use of PCR arrays, gene expression of cytokines (Csf1, Csf3), chemokines (Ccl1, Ccl2, Ccl6, Ccl8, Ccl11, Ccl12, Cxcl4, Cxcl9, Cxcl12), and their receptors (Ccr1, Ccr2, Ccr3, Ccr5) were also reduced significantly in Het compared with pregnant WT uteri. Moreover, with the use of flow cytometry, myeloid CSF1R and CCR2 expression in blood and uteri from both pregnant and nonpregnant mice was characterized, and a deficiency in HO-1 significantly reduced CCR2 expression in infiltrating uterine monocytes/macrophages and dendritic cells (DCs). These data reveal that HO-1 regulates not only cytokine/chemokine production in pregnant uteri but also myeloid cell receptor numbers, suggesting a role of HO-1 in the recruitment and maintenance of myeloid cells in pregnant uteri and subsequent effects on placental vascular formation., (© Society for Leukocyte Biology.)

Background: Severe hemolytic disease of the newborn leads to the release of pro-oxidative free heme (FH). Heme oxygenase (HO) is primarily responsible for detoxifying FH., Objective: To investigate the protective effects of HO in a model of heme overload., Methods: For in vitro studies, NIH3T3 HO-1-luc cells were incubated with 10, 30, or 60 µM FH or methemalbumin (MHA). HO-1 promoter activity was assessed 3, 6, and 24 h after treatment. Cell survival was indexed by viability assays. For in vivo studies, 1- and 5-week-old wild-type (Wt) or HO-1-heterozygous (Het, HO-1+/-) mice were given 60 µmol FH or MHA/kg intraperitoneally. After 24 h, plasma aspartate aminotransferease (AST)/alanine transaminase (ALT) and hemopexin, liver HO activity, and lipid peroxidation (LP) were determined., Results: In HO-1-luc cells, HO-1 promoter activity peaked 6 h after incubation with 30 µM FH (1.6-fold) or 60 µM MHA (2.1-fold) over baseline. Twenty-four hours after exposure to 60 µM FH, a decrease in viability of 80% was found, compared with no decrease after exposure to 60 µM MHA. In 1-week-old Wt and HO-1 Het pups given 60 µmol FH/kg, HO activity significantly increased 3.5- and 3.1-fold, respectively. No changes in LP or AST/ALT levels were observed. In adult Wt and HO-1 Het mice, HO activity increased (3.0- and 2.6-fold, respectively). LP and AST levels significantly increased 28.4- and 2.7-fold, respectively, in adult HO-1 Het mice. Hemopexin levels at baseline were higher in adults compared with newborns for both Wt and Het mice. In addition, FH induced hemopexin levels in both adults and newborns, but to a lesser degree in newborns., Conclusions: FH is highly toxic in vitro, but its toxicity is abolished when bound to albumin. Newborns appear to be protected from the pro-oxidative effects of FH, which may be mediated by heme binding and a higher absolute HO activity at baseline and after FH-mediated induction., (© 2017 S. Karger AG, Basel.)

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, is a cytoprotective enzyme upregulated in the vasculature by increased flow and inflammatory stimuli. Human genetic data suggest that a diminished HO-1 expression may predispose one to abdominal aortic aneurysm (AAA) development. In addition, heme is known to strongly induce HO-1 expression. Utilizing the porcine pancreatic elastase (PPE) model of AAA induction in HO-1 heterozygous (HO-1+/-, HO-1 Het) mice, we found that a deficiency in HO-1 leads to augmented AAA development. Peritoneal macrophages from HO-1+/- mice showed increased gene expression of pro-inflammatory cytokines, including MCP-1, TNF-alpha, IL-1-beta, and IL-6, but decreased expression of anti-inflammatory cytokines IL-10 and TGF-beta. Furthermore, treatment with heme returned AAA progression in HO-1 Het mice to a wild-type profile. Using a second murine AAA model (Ang II-ApoE-/-), we showed that low doses of the HMG-CoA reductase inhibitor rosuvastatin can induce HO-1 expression in aortic tissue and suppress AAA progression in the absence of lipid lowering. Our results support those studies that suggest that pleiotropic statin effects might be beneficial in AAA, possibly through the upregulation of HO-1. Specific targeted therapies designed to induce HO-1 could become an adjunctive therapeutic strategy for the prevention of AAA disease.

Background: Increased bilirubin production due to hemolysis can lead to neonatal hyperbilirubinemia. Inhibition of heme oxygenase (HO), the rate-limiting enzyme in heme catabolism, by metalloporphyrins (Mps) may be an ideal preventive strategy for neonatal hemolytic disease. Zinc protoporphyrin (ZnPP) is a naturally occurring Mp, potent, not phototoxic, with minimal HO-1 upregulation, but is not orally absorbed. Recently, we designed a lipid-based ZnPP formulation (ZnPP-Lipid), which is orally absorbed by newborn mice. Here, we evaluated the efficacy of ZnPP-Lipid in heme-loaded newborn mice, a model analogous to hemolytic infants., Methods: After 24 h of heme administration (30 µmol/kg s.c.), 4-d-old mice were given 30 µmol ZnPP-Lipid/kg via intragastric injections. After 3 h, liver and brain HO activity were measured. HO-1 upregulation was assessed by determinations of HO-1 protein, promoter activity, and mRNA by Western blot, in vivo bioluminescence imaging, and RT-PCR, respectively., Results: After heme loading, liver HO activity significantly increased ~1.6-fold, which was inhibited in a dose-dependent manner by ZnPP-Lipid. A dose of 30 µmol/kg returned activity to control levels. Brain HO activity was not inhibited. No significant increases in liver and brain HO-1 protein, promoter activity, and mRNA were observed., Conclusion: ZnPP-Lipid is effective and thus has potential for treating neonatal hyperbilirubinemia due to hemolysis.

Background: Necrotizing enterocolitis (NEC), an intestinal inflammatory disease affecting premature infants, is associated with low regulatory T (Treg) to effector T (Teff) cell ratios. We recently demonstrated that heme oxygenase-1 (HO-1) deficiency leads to increased NEC development. Here, we investigated the effects of HO-1 on T-cell proportions in a murine NEC-like injury model., Methods: Intestinal injury was induced in 7-d-old wild-type (WT) or HO-1 heterozygous (HO-1 Het) pups by formula-feeding every 4 h for 24-78 h by oral gavage and exposures to 5%O2. Controls remained breastfed. HO-1 was induced in WT pups by administering heme preinjury induction. Lamina propria T cells were identified by flow cytometry. For adoptive transfer studies, WT splenic/thymic Tregs were injected intraperitoneally into HO-1 Het pups 12-24 h preinduction., Results: Het mice showed increased intestinal injury and decreased Treg/Teff ratios. Genes for pattern recognition (Toll-like receptor-4, C-reactive protein, MyD88) and neutrophil recruitment increased in Het pups after NEC induction. Inducing intestinal HO-1 decreased NEC scores and incidence, and increased Treg/Teff ratios. Moreover, adoptive transfer of Tregs from WT to HO-1 Het pups decreased NEC scores and incidence and restored Treg/Teff ratios., Conclusion: HO-1 can change Treg proportions in the lamina propria of young mice under inflammatory conditions, which might, in part, confer intestinal protection.

Leukocyte infiltration into the uterus is a characteristic feature in early to midpregnancy, but the composition and function of these leukocytes are not well understood. Using a pregnant murine model, we showed that myeloid cells and uterine NK (uNK) cells were the predominant populations in uteri during early to midgestation, whereas T and B cells were constrained. Uterine myeloid populations included cells that infiltrated from the circulation (myeloid-derived suppressor cells [MDSCs], monocyte-derived macrophages [Mφs], and dendritic cells [DCs]) or proliferated from resident precursors (resident Mφs [Re-Mφs] and DCs). CD11b(hi)Ly6-G(hi) cells, representing neutrophils in both blood and uterine MDSCs, significantly increased from embryonic days 8.5 to 9.5. To understand their putative functions, we used anti-Gr-1 Ab to deplete circulating neutrophils and uterine MDSCs. In the absence of MDSC suppression, uterine DCs, T cells, and regulatory T cells expanded. Conversely, uterine MDSCs responded to LPS-induced inflammation and transformed into CD14(+)-activated neutrophils, resulting in an upregulation of tolerogenic DCs. A high dose of LPS (2.5 μg/mouse) significantly increased the influx of neutrophils and production of proinflammatory cytokines, such as IL-1β and TNF-α, resulting in the reduction of Re-Mφs and uNK cells, and led to placental hemorrhages and fetal deaths. In summary, uterine MDSCs are important in early to midpregnancy by responding to the maternal immunologic milieu and protecting uNK cells and Re-Mφs via MDSC's suppressive and anti-inflammatory functions. Upsetting this delicate immune balance by factors leading to either insufficient MDSCs or excessive neutrophil infiltration in the fetomaternal interface may contribute to pregnancy failure., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Necrotizing enterocolitis (NEC) is typified by mucosal destruction, which subsequently can lead to intestinal necrosis. Prematurity, enteral feeding, and bacterial colonization are the main risk factors and, combined with other stressors, can cause increased intestinal permeability, injury, and an exaggerated inflammatory response. Heme oxygenase-1 (HO-1) mediates intestinal protection due to anti-inflammatory, antioxidative, and antiapoptotic effects of its products carbon monoxide, biliverdin, and bilirubin. This study investigates a possible role of HO-1 in the pathogenesis of NEC using a newborn mouse model. We induced NEC-like intestinal injury in 7-day-old HO-1 heterozygous (HO-1 Het, Hmox1(+/-)) and wild-type (Wt, Hmox1(+/+)) mice by gavage feeding and hypoxic exposures. Control (Con) pups of both genotypes were dam-fed. Intestines of HO-1 Het Con pups appeared predisposed to injury, with higher histological damage scores, more TUNEL-positive cells, and a significant reduction in muscularis externa thickness compared with Wt Con pups. The increase in HO activity after HO-1 induction by the substrate heme or by hypoxic stress was significantly impaired in HO-1 Het pups. After induction of intestinal injury, HO-1 Het pups displayed significantly higher NEC incidence (78 vs. 43%), mortality (83 vs. 54%), and median scores (2.5 vs. 1.5) than Wt NEC pups. PCR array analyses revealed increased expressions of IL-1β, P-selectin, matrix metallopeptidase 2, collagen type XVIII-α1, serpine 1, and others in NEC-induced HO-1 Het ileal and jejunal tissues. We conclude that a partial HO-1 deficiency promotes experimental NEC-like intestinal injury, possibly mediated by exaggerated inflammation and disruption in tissue repair.

The placental vasculature is critical for nutrient, gas, and waste exchange between the maternal and fetal systems. Its development depends on the proper expression and interaction of angiogenesis and associated growth factors. Heme oxygenase (HMOX), the enzyme for heme degradation, plays a role in angiogenesis and is highly expressed in the placenta. To evaluate the role of maternal HMOX1, the inducible HMOX isozyme, on placental vasculature formation, mice with a partial deficiency in Hmox1 (Hmox1(+/-)) were used. Three-dimensional images of placental vasculatures as well as spiral arteries from Hmox1(+/+) or Hmox1(+/-) placentas were created by vascular corrosion casting technique and imaged by micro-computerized tomography (microCT). The structures and morphologies of fetomaternal interfaces were observed by histological staining and the ultrastructure of uterine natural killer (uNK) cells, a major regulator in spiral artery remodeling, was analyzed by transmission electron microscopy. A group of growth factors and angiogenic factors from the decidua/mesometrial lymphoid aggregate of pregnancy (MLAp) as well as labyrinth regions were quantified using an angiogenesis PCR array kit and compared between Hmox1(+/+) or Hmox1(+/-) placentas. In conclusion, a partial deficiency of maternal Hmox1 resulted in the malformation of fetomaternal interface, insufficiency of spiral artery remodeling, and alteration of uNK cell differentiation and maturation. These changes were independent of the fetal genotype, but relied on the maternal HMOX1 level, which determined the balance of expression levels of pro- and antiangiogenic factors in the decidua/MLAp region. These results implied that Hmox1 polymorphisms among the human population might contribute to some unexplained cases of pregnancy disorders, such as fetal growth retardation and preeclampsia.

The cytoprotective response to thermal injury is characterized by transcriptional activation of "heat shock proteins" (hsp) and proinflammatory proteins. Expression of these proteins may predict cellular survival. Microarray analyses were performed to identify spatially distinct gene expression patterns responding to thermal injury. Laser injury zones were identified by expression of a transgene reporter comprised of the 70 kD hsp gene and the firefly luciferase coding sequence. Zones included the laser spot, the surrounding region where hsp70-luc expression was increased, and a region adjacent to the surrounding region. A total of 145 genes were up-regulated in the laser irradiated region, while 69 were up-regulated in the adjacent region. At 7 hours the chemokine Cxcl3 was the highest expressed gene in the laser spot (24 fold) and adjacent region (32 fold). Chemokines were the most common up-regulated genes identified. Microarray gene expression was successfully validated using qRT- polymerase chain reaction for selected genes of interest. The early response genes are likely involved in cytoprotection and initiation of the healing response. Their regulatory elements will benefit creating the next generation reporter mice and controlling expression of therapeutic proteins. The identified genes serve as drug development targets that may prevent acute tissue damage and accelerate healing.

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 constituitive 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), the rate-limiting enzyme in heme degradation, plays a role in neonatal jaundice. Understanding the regulation of the developmental expression patterns of the two HO isozymes, HO-1 and HO-2, is essential for targeting HO to control pathologic jaundice, and uncovering the fundamental role that they play in mammalian development. Here we characterized the ontogeny of HO-1 and HO-2 expression in the developing mouse cortex by in vivo bioluminescence imaging, quantitative RT-PCR, and Western blot. HO-2, the predominant isoform in the adult cortex, was relatively stable throughout all ages. HO-1 was observed to be progressively down-regulated in an age-related manner. HO-1 expression in the adult cortex was also the lowest among the eight adult tissues analyzed. Because there is a 283-bp CpG island region in the HO-1 promoter, we hypothesized that methylation of the island is responsible for the age-related HO-1 down-regulation in the cortex. Methylation status was assessed using regular and quantitative methylation-specific PCR and the CpG island was found to be hypomethylated at all ages. Therefore, we conclude that HO-1 gene expression in the cortex is developmentally-regulated and that methylation of the HO-1 CpG island is not associated with the down-regulation of the gene.

In vivo bioluminescence imaging depends on light emitted by luciferases in the body overcoming the effect of tissue attenuation. Understanding this relationship is essential for detection and quantification of signal. We have studied four codon optimized luciferases with different emission spectra, including enzymes from firefly (FLuc), click beetle (CBGr68, CBRed) and Renilla reniformins (hRLuc). At 25 degrees C, the in vitro lambda(max) of these reporters are 578, 543, 615, and 480 nm, respectively; at body temperature, 37 degrees C, the brightness increases and the firefly enzyme demonstrates a 34-nm spectral red shift. Spectral shifts and attenuation due to tissue effects were evaluated using a series of 20-nm bandpass filters and a cooled charge-coupled device (CCD) camera. Attenuation increased and the spectra of emitted light was red shifted for signals originating from deeper within the body relative to superficial origins. The tissue attenuation of signals from CBGr68 and hRLuc was greater than from those of Fluc and CBRed. To further probe tissue effects, broad spectral emitters were created through gene fusions between CBGr68 and CBRed. These resulted in enzymes with broader emission spectra, featuring two peaks whose intensities are differentially affected by temperature and tissue depth. These spectral measurement data allow for improved understanding of how these reporters can be used in vivo and what they can reveal about biological processes in living subjects.