Naoko Brown

Substrate-selective COX-2 inhibition decreases anxiety via endocannabinoid activation

Augmentation of endogenous cannabinoid (eCB) signaling represents an emerging approach to the treatment of affective disorders. Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid to form prostaglandins, but also inactivates eCBs in vitro. However, the viability of COX-2 as a therapeutic target for in vivo eCB augmentation has not been explored. Using medicinal chemistry and in vivo analytical and behavioral pharmacological approaches, we found that COX-2 is important for the regulation of eCB levels in vivo. We used a pharmacological strategy involving substrate-selective inhibition of COX-2 to augment eCB signaling without affecting related non-eCB lipids or prostaglandin synthesis. Behaviorally, substrate-selective inhibition of COX-2 reduced anxiety-like behaviors in mice via increased eCB signaling. Our data suggest a key role for COX-2 in the regulation of eCB signaling and indicate that substrate-selective pharmacology represents a viable approach for eCB augmentation with broad therapeutic potential.

COX-2 compensation in the uterus of COX-1 deficient mice during the pre-implantation period.

Prostaglandins (PGs) produced by cyclooxygenase (COX) participate in many aspects of female reproduction. The two isoforms of cyclooxygenase, COX-1 and COX-2, have distinct expression patterns in the mouse uterus during the peri-implantation period and suggest their independent contribution to uterine PGs. Using wild type and COX-1(-/-) mice, we examined the role of COX-1-derived PGs on day 4 of pregnancy, when its expression is maximal. Uterine vascular permeability was measured by 125I-labeled bovine serum albumin (BSA) uptake, and PG content was measured by gas chromatography-mass spectrometry. Vascular permeability and PG concentrations were reduced in COX-1(-/-) mice, but by less than the expected amount. After ovariectomy, uterine vascular permeability declined in both groups, but returned to baseline in wild type and was exaggerated in COX-1(-/-) females after treatment with ovarian steroids. Most importantly, COX-1(-/-) uteri displayed COX-2 expression on the morning of day 4, when COX-2 is normally absent. This hybridization pattern resembles the native expression of COX-1, and may partially offset the loss of COX-1-derived PGs. These data indicate that COX-1-derived PGs are important during uterine preparation for implantation, and that COX-2 compensation occurs in the absence of COX-1.

Zonula occludens-1 (ZO-1) is involved in morula to blastocyst transformation in the mouse

It is unknown whether or not tight junction formation plays any role in morula to blastocyst transformation that is associated with development of polarized trophoblast cells and fluid accumulation. Tight junctions are a hallmark of polarized epithelial cells and zonula occludens-1 (ZO-1) is a known key regulator of tight junction formation. Here we show that ZO-1 protein is first expressed during compaction of 8-cell embryos. This stage-specific appearance of ZO-1 suggests its participation in morula to blastocyst transition. Consistent with this idea, we demonstrate that ZO-1 siRNA delivery inside the blastomeres of zona-weakened embryos using electroporation not only knocks down ZO-1 gene and protein expressions, but also inhibits morula to blastocyst transformation in a concentration-dependent manner. In addition, ZO-1 inactivation reduced the expression of Cdx2 and Oct-4, but not ZO-2 and F-actin. These results provide the first evidence that ZO-1 is involved in blastocyst formation from the morula by regulating accumulation of fluid and differentiation of nonpolar blastomeres to polar trophoblast cells.

Efficacy of paracetamol on patent ductus arteriosus closure may be dose dependent: evidence from human and murine studies

Background:We evaluated the clinical effectiveness of variable courses of paracetamol on patent ductus arteriosus (PDA) closure and examined its effect on the in vitro term and preterm murine ductus arteriosus (DA).Methods:Neonates received one of the following three paracetamol regimens: short course of oral paracetamol (SCOP), long course of oral paracetamol (LCOP), and intravenous paracetamol (IVP) for 2–6 d. Pressure myography was used to examine changes in vasomotor tone of the preterm and term mouse DA in response to paracetamol or indomethacin. Their effect on prostaglandin synthesis by DA explants was measured by mass spectroscopy.Results:Twenty-one preterm infants were included. No changes in PDA hemodynamics were seen in SCOP infants (n = 5). The PDA became less significant and eventually closed in six LCOP infants (n = 7). PDA closure was achieved in eight IVP infants (n = 9). On pressure myograph, paracetamol induced a concentration-dependent constriction of the term mouse DA, up to 30% of baseline (P < 0.01), but required >1 µmol/l. Indomethacin induced greater DA constriction and suppression of prostaglandin synthesis (P < 0.05).Conclusion:The clinical efficacy of paracetamol on PDA closure may depend on the duration of treatment and the mode of administration. Paracetamol is less potent than indomethacin for constriction of the mouse DA in vitro.Pediatric Research (2014); 76 3, 238–244. doi:10.1038/pr.2014.82

Embryo-Uterine Interactions via the Neuregulin Family of Growth Factors During Implantation in the Mouse

Abstract Neuregulins (NRGs) are cell-signaling molecules with recognized roles in cancer and development, but little is known about their role in embryo implantation. Among representative NRG-1 isoforms, neu differentiation factor (NDF, type I) is expressed in the female reproductive tract and is localized to the implantation site. Here, we show that sensory and motor neuron-derived factor (SMDF, type III) is expressed in the uterine subepithelial stroma around the blastocyst and is only upregulated at the time of implantation. The cellular distribution of SMDF is similar to that of NDF and requires an implantation-competent blastocyst. The glial growth factor (GGF, type II) isoform of NRG-1 and the NRG-2 and NRG-3 genes were not expressed in the peri-implantation uterus, as determined by reverse transcription-polymerase chain reaction or in situ hybridization. In contrast to the cellular expression pattern of NDF and SMDF, NRG-4 was present in the luminal and glandular epithelium throughout the uterus during the preimplantation period. Expression of NRG-4 declined in the uterine luminal epithelium during implantation but persisted in the glandular epithelium through Day 8 of pregnancy. Studies in ovariectomized mice showed that NRG-4 is a progesterone-regulated gene, with partial augmentation by estrogen. We also observed upregulation of the erbB2 and erbB3 receptors at the blastocyst stage of embryo development. Together, these findings suggest that a distinct subset of NRGs participates in the signaling network that directs embryo implantation. Upregulation of embryonic erbB2/ erbB3 in the blastocyst trophectoderm and induction of certain NRG-1 isoforms with blastocyst activation help to define additional aspects of the embryo-uterine cross-talk that underlies the implantation process.

Regulation of the fetal mouse ductus arteriosus is dependent on interaction of nitric oxide and COX enzymes in the ductal wall

Nitric oxide (NO) and cyclooxygenase (COX)-derived prostaglandins are critical regulators of the fetal ductus arteriosus. To examine the interaction of these pathways within the ductus wall, the ductus arteriosus of term and preterm fetal mice was evaluated by pressurized myography. The isolated preterm ductus was more sensitive to NOS inhibition than at term. Sequential NOS and COX inhibition caused 36% constriction of the preterm ductus regardless of drug order. In contrast, constriction of the term ductus was dependent on the sequence of inhibition; NOS inhibition prior to COX inhibition produced greater constriction than when inhibitors were given in reverse order (36+/-6% versus 23+/-5%). Selective COX-1 or COX-2 inhibition prior to N(G)-nitro-l-arginine methyl ester (l-NAME) induced the expected degree of constriction. However, NOS inhibition followed by selective COX-2 inhibition caused unexpected ductal dilation. These findings are consistent with NO-induced activation of COX in the ductus arteriosus wall and the production of a COX-2-derived constrictor prostanoid that contributes to the balance of vasoactive forces that maintain fetal ductus arteriosus tone.

Neurosensory perception of environmental cues modulates sperm motility critical for fertilization.

Scents and Sperm Once sperm enter the female reproductive tract, they have an arduous task to find an egg at a distant, often concealed, location. McKnight et al. (p. 754) show that Caenorhabditis elegans make this task more or less difficult, depending on pheromones in the external environment. Pheromones perceived by female sensory neurons modulate the synthesis of ovarian prostaglandins, which provide sperm positional information. Thus, environmental cues can indirectly impact sperm function even when the sperm themselves are not directly exposed. Nematode pheromones modulate a neuroendocrine pathway that converts dietary fats into sperm-attracting prostaglandins. Environmental exposures affect gamete function and fertility, but the mechanisms are poorly understood. Here, we show that pheromones sensed by ciliated neurons in the Caenorhabditis elegans nose alter the lipid microenvironment within the oviduct, thereby affecting sperm motility. In favorable environments, pheromone-responsive sensory neurons secrete a transforming growth factor–β ligand called DAF-7, which acts as a neuroendocrine factor that stimulates prostaglandin-endoperoxide synthase [cyclooxygenase (Cox)]–independent prostaglandin synthesis in the ovary. Oocytes secrete F-class prostaglandins that guide sperm toward them. These prostaglandins are also synthesized in Cox knockout mice, raising the possibility that similar mechanisms exist in other animals. Our data indicate that environmental cues perceived by the female nervous system affect sperm function.

Detecting Biochemical Changes in the Rodent Cervix During Pregnancy Using Raman Spectroscopy

The goal of this research is to determine whether Raman spectroscopy (RS), an optical method that probes the vibrational modes of tissue components, can be used in vivo to study changes in the mouse cervix during pregnancy. If successful, such a tool could be used to detect cervical changes due to pregnancy, both normal and abnormal, in animal models and humans. For this study, Raman spectra were acquired before, during and after a 19-day mouse gestational period. In some cases, after Raman data was obtained, cervices were excised for structural testing and histological staining for collagen and smooth muscle. Various peaks of the Raman spectra, such as the areas corresponding to fatty acid content and collagen organization, changed as the cervix became softer in preparation for labor and delivery. These findings correspond to the increase in compliance of the tissue and the collagen disorganization visualized with the histological staining. The results of this study suggest that non-invasive RS can be used to study cervical changes during pregnancy, labor and delivery and can possibly predict preterm delivery before overt clinical manifestations, potentially lead to more effective preventive and therapeutic interventions.

Isoprostanes as physiological mediators of transition to newborn life: novel mechanisms regulating patency of the term and preterm ductus arteriosus

Background:Increased oxygen tension at birth regulates physiologic events that are essential to postnatal survival, but the accompanying oxidative stress may also generate isoprostanes. We hypothesized that isoprostanes regulate ductus arteriosus (DA) function during postnatal vascular transition.Methods:Isoprostanes were measured by gas chromatography–mass spectrometry. DA tone was assessed by pressure myography. Gene expression was measured by quantitative PCR.Results:Oxygen exposure was associated with increased 8-iso-prostaglandin (PG)F2α in newborn mouse lungs. Both 8-iso-PGE2 and 8-iso-PGF2α induced concentration-dependent constriction of the isolated term DA, which was reversed by the thromboxane A2 (TxA2) receptor antagonist SQ29548. SQ29548 pretreatment unmasked an isoprostane-induced DA dilation mediated by the EP4 PG receptor. Exposure of the preterm DA to 8-iso-PGE2 caused unexpected DA relaxation that was reversed by EP4 antagonism. In contrast, exposure to 8-iso-PGF2α caused preterm DA constriction via TxA2 receptor activation. Further investigation revealed the predominance of the TxA2 receptor at term, whereas the EP4 receptor was expressed and functionally active from mid-gestation onward.Conclusion:This study identifies a novel physiological role for isoprostanes during postnatal vascular transition and provide evidence that oxidative stress may act on membrane lipids to produce vasoactive mediators that stimulate physiological DA closure at birth or induce pathological patency of the preterm DA.

Transcriptional profiling reveals ductus arteriosus-specific genes that regulate vascular tone

Failure of the ductus arteriosus (DA) to close at birth can lead to serious complications. Conversely, certain profound congenital cardiac malformations require the DA to be patent until corrective surgery can be performed. In each instance, clinicians have a very limited repertoire of therapeutic options at their disposal - indomethacin or ibuprofen to close a patent DA (PDA) and prostaglandin E1 to maintain patency of the DA. Neither treatment is specific to the DA and both may have deleterious off-target effects. Therefore, more therapeutic options specifically targeted to the DA should be considered. We hypothesized the DA possesses a unique genetic signature that would set it apart from other vessels. A microarray was used to compare the genetic profiles of the murine DA and ascending aorta (AO). Over 4,000 genes were differentially expressed between these vessels including a subset of ion channel-related genes. Specifically, the alpha and beta subunits of large-conductance calcium-activated potassium (BKCa) channels are enriched in the DA. Gain- and loss-of-function studies showed inhibition of BKCa channels caused the DA to constrict, while activation caused DA relaxation even in the presence of O2. This study identifies subsets of genes that are enriched in the DA that may be used to develop DA-specific drugs. Ion channels that regulate DA tone, including BKCa channels, are promising targets. Specifically, BKCa channel agonists like NS1619 maintain DA patency even in the presence of O2 and may be clinically useful.