Discerning the Role of Prostaglandins in Ductus Arteriosus Remodeling

The ductus arteriosus (DA) is a fetal pulmonary bypass shunt that constricts and permanently remodels during the transition from fetal to adult circulation. Prostaglandin E2 (PGE2) is potent mediator of numerous physiological responses both in homeostasis and disease state. PGE2 play a vital role in DA maturation and closure, although the exact molecular role is unclear. We attempt to discern the nature of PGE2 involvement in DA maturation and closure. Here we generate a conditional null allele of the prostaglandin E receptor 4 (EP4), which has been previously shown to be responsible for PGE2 signaling in the DA. Utilizing various tissue specific Cre recombinase transgenes, we have shown that EP4 expression on the neural crest derived smooth muscle cells of the DA is critical for proper DA closure. We have also shown that endothelial expression of the PGE2 vasodilatory receptors (EP4 and EP2) is non-essential for DA closure or vascular development. Genome wide expression profiling of the wildtype DA and EP4 deficient DA were used to assess the transcriptional consequences of PGE2/EP4 signaling in the DA. Differentially expressed genes in the wildtype DA indicate that EP4 receptor expression leads to the up-regulation of numerous cytoskeletal genes. The relative minor increase (<2 fold) of numerous cytoskeletal genes may explain why the wildtype and EP4 deficient DA appear morphologically similar in utero but have antithetical fates after birth. Here we also document the existence of a prostaglandin-independent mechanism of DA maturation and closure in mice. Selective mating generated a recombinant inbred (RI) mouse strain that undergoes DA maturation and closure without any contribution from prostaglandin signaling. Single locus inheritance of patent ductus arteriosus (PDA) in common inbred strains (CIS) seemed at odds with the complex inheritance pattern of PDA in larger animals, but the RI strain indicates that DA closure in non-CIS mice is also a complex trait. The study of both prostaglandin-dependent DA closure of CIS mice and prostaglandin-independent DA closure of RI mice provides a mouse model for understanding the complex trait of larger animals

PGE2 through the EP4 receptor controls smooth muscle gene expression patterns in the ductus arteriosus critical for remodeling at birth

The ductus arteriosus (DA) is a fetal shunt that directs right ventricular outflow away from pulmonary circulation and into the aorta. Critical roles for prostaglandin E2 (PGE2) and the EP4 receptor (EP4) have been established in maintaining both the patency of the vessel in utero and in its closure at birth. Here we have generated mice in which loss of EP4 expression is limited to either the smooth muscle (SMC) or endothelial cells and demonstrated that SMC, but not endothelial cell expression of EP4 is required for DA closure. The genome wide expression analysis of full term wild type and EP4−/− DA indicates that PGE2/EP4 signaling modulates expression of a number of unique pathways, including those involved in SMC proliferation, cell migration, and vascular tone. Together this supports a mechanism by which maturation and increased contractility of the vessel is coupled to the potent smooth muscle dilatory actions of PGE2

Chromatin architectural factor CTCF is essential for progesterone-dependent uterine maturation

Receptors for estrogen and progesterone frequently interact, via Cohesin/CTCF loop extrusion, at enhancers distal from regulated genes. Loss-of-function CTCF mutation in &gt;20% of human endometrial tumors indicates its importance in uterine homeostasis. To better understand how CTCF-mediated enhancer-gene interactions impact endometrial development and function, the Ctcf gene was selectively deleted in female reproductive tissues of mice. Prepubertal Ctcfd/d uterine tissue exhibited a marked reduction in the number of uterine glands compared to those without Ctcf deletion (Ctcff/f mice). Post-pubertal Ctcfd/d uteri were hypoplastic with significant reduction in both the amount of the endometrial stroma and number of glands. Transcriptional profiling revealed increased expression of stem cell molecules Lif, EOMES, and Lgr5, and enhanced inflammation pathways following Ctcf deletion. Analysis of the response of the uterus to steroid hormone stimulation showed that CTCF deletion affects a subset of progesterone-responsive genes. This finding indicates (1) Progesterone-mediated signaling remains functional following Ctcf deletion and (2) certain progesterone-regulated genes are sensitive to Ctcf deletion, suggesting they depend on gene-enhancer interactions that require CTCF. The progesterone-responsive genes altered by CTCF ablation included Ihh, Fst, and Errfi1. CTCF-dependent progesterone-responsive uterine genes enhance critical processes including anti-tumorigenesis, which is relevant to the known effectiveness of progesterone in inhibiting progression of early-stage endometrial tumors. Overall, our findings reveal that uterine Ctcf plays a key role in progesterone-dependent expression of uterine genes underlying optimal post-pubertal uterine development.</p

Generation and characterization of epoxide hydrolase 3 (EPHX3)-deficient mice.

Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs), which play an important role in blood pressure regulation, protection against ischemia-reperfusion injury, angiogenesis, and inflammation. Epoxide hydrolases metabolize EETs to their corresponding diols (dihydroxyeicosatrienoic acids; DHETs) which are biologically less active. Microsomal epoxide hydrolase (EPHX1, mEH) and soluble epoxide hydrolase (EPHX2, sEH) were identified >30 years ago and are capable of hydrolyzing EETs to DHETs. A novel epoxide hydrolase, EPHX3, was recently identified by sequence homology and also exhibits epoxide hydrolase activity in vitro with a substrate preference for 9,10-epoxyoctadecamonoenoic acid (EpOME) and 11,12-EET. EPHX3 is highly expressed in the skin, lung, stomach, esophagus, and tongue; however, its endogenous function is unknown. Therefore, we investigated the impact of genetic disruption of Ephx3 on fatty acid epoxide hydrolysis and EET-related physiology in mice. Ephx3-/- mice were generated by excising the promoter and first four exons of the Ephx3 gene using Cre-LoxP methodology. LC-MS/MS analysis of Ephx3-/- heart, lung, and skin lysates revealed no differences in endogenous epoxide:diol ratios compared to wild type (WT). Ephx3-/- mice also exhibited no change in plasma levels of fatty acid epoxides and diols relative to WT. Incubations of cytosolic and microsomal fractions prepared from Ephx3-/- and WT stomach, lung, and skin with synthetic 8,9-EET, 11,12-EET, and 9,10-EpOME revealed no significant differences in rates of fatty acid diol formation between the genotypes. Ephx3-/- hearts had similar functional recovery compared to WT hearts following ischemia/reperfusion injury. Following intranasal lipopolysaccharide (LPS) exposure, Ephx3-/- mice were not different from WT in terms of lung histology, bronchoalveolar lavage fluid cell counts, or fatty acid epoxide and diol levels. We conclude that genetic disruption of Ephx3 does not result in an overt phenotype and has no significant effects on the metabolism of EETs or EpOMEs in vivo

EH3 (ABHD9): the first member of a new epoxide hydrolase family with high activity for fatty acid epoxides

Epoxide hydrolases are a small superfamily of enzymes important for the detoxification of chemically reactive xenobiotic epoxides and for the processing of endogenous epoxides that act as signaling molecules. Here, we report the identification of two human epoxide hydrolases: EH3 and EH4. They share 45% sequence identity, thus representing a new family of mammalian epoxide hydrolases. Quantitative RT-PCR from mouse tissue indicates strongest EH3 expression in lung, skin, and upper gastrointestinal tract. The recombinant enzyme shows a high turnover number with 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid (EET), as well as 9,10-epoxyoctadec-11-enoic acid (leukotoxin). It is inhibited by a subclass of N,N'-disubstituted urea derivatives, including 12-(3-adamantan-1-yl-ureido)-dodecanoic acid, 1-cyclohexyl-3-dodecylurea, and 1-(1-acetylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea, compounds so far believed to be selective inhibitors of mammalian soluble epoxide hydrolase (sEH). Its sensitivity to this subset of sEH inhibitors may have implications on the pharmacologic profile of these compounds. This is particularly relevant because sEH is a potential drug target, and clinical trials are under way exploring the value of sEH inhibitors in the treatment of hypertension and diabetes type II

Podocyte-specific soluble epoxide hydrolase deficiency in mice attenuates acute kidney injury.

Podocytes play an important role in maintaining glomerular function, and podocyte injury is a significant component in the pathogenesis of proteinuria. Soluble epoxide hydrolase (sEH) is a cytosolic enzyme whose genetic deficiency and pharmacological inhibition have beneficial effects on renal function, but its role in podocytes remains unexplored. The objective of this study was to investigate the contribution of sEH in podocytes to lipopolysaccharide (LPS)-induced kidney injury. We report increased sEH transcript and protein expression in murine podocytes upon LPS challenge. To determine the function of sEH in podocytes in&nbsp;vivo we generated podocyte-specific sEH-deficient (pod-sEHKO) mice. Following LPS challenge, podocyte sEH-deficient mice exhibited lower kidney injury, proteinuria, and blood urea nitrogen concentrations than controls suggestive of preserved renal function. Also, renal mRNA and serum concentrations of inflammatory cytokines IL-6, IL-1β, and TNFα were significantly lower in LPS-treated pod-sEHKO than control mice. Moreover, podocyte sEH deficiency was associated with decreased LPS-induced NF-κB and MAPK activation and attenuated endoplasmic reticulum stress. Furthermore, the protective effects of podocyte sEH deficiency in&nbsp;vivo were recapitulated in E11 murine podocytes treated with a selective sEH pharmacological inhibitor. Altogether, these findings identify sEH in podocytes as a contributor to signaling events in acute renal injury and suggest that sEH inhibition may be of therapeutic value in proteinuria.EnzymesSoluble epoxide hydrolase: EC 3.3.2.10