Effects of sEH inhibition on the eicosanoid and cytokine storms in SARS-CoV-2-infected mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection involves an initial viral infection phase followed by a host-response phase that includes an eicosanoid and cytokine storm, lung inflammation and respiratory failure. While vaccination and early anti-viral therapies are effective in preventing or limiting the pathogenic host response, this latter phase is poorly understood with no highly effective treatment options. Inhibitors of soluble epoxide hydrolase (sEH) increase levels of anti-inflammatory molecules called epoxyeicosatrienoic acids (EETs). This study aimed to investigate the impact of sEH inhibition on the host response to SARS-CoV-2 infection in a mouse model with human angiotensin-converting enzyme 2 (ACE2) expression. Mice were infected with SARS-CoV-2 and treated with either vehicle or the sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU). At day 5 post-infection, SARS-CoV-2 induced weight loss, clinical signs, a cytokine storm, an eicosanoid storm, and severe lung inflammation with ~50% mortality on days 6-8 post-infection. SARS-CoV-2 infection induced lung expression of phospholipase A2 (PLA2), cyclooxygenase (COX) and lipoxygenase (LOX) pathway genes, while suppressing expression of most cytochrome P450 genes. Treatment with the sEH inhibitor TPPU delayed weight loss but did not alter clinical signs, lung cytokine expression or overall survival of infected mice. Interestingly, TPPU treatment significantly reversed the eicosanoid storm and attenuated viral-induced elevation of 39 fatty acids and oxylipins from COX, LOX and P450 pathways, which suggests the effects at the level of PLA2 activation. The suppression of the eicosanoid storm by TPPU without corresponding changes in lung cytokines, lung inflammation or mortality reveals a surprising dissociation between systemic oxylipin and cytokine signaling pathways during SARS-CoV-2 infection and suggests that the cytokine storm is primarily responsible for morbidity and mortality in this animal model.

Disruption of Ephx2 in cardiomyocytes but not endothelial cells improves functional recovery after ischemia-reperfusion in isolated mouse hearts.

Cytochromes P450 metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which have numerous effects. After cardiac ischemia, EET-induced coronary vasodilation increases delivery of oxygen/nutrients to the myocardium, and EET-induced signaling protects cardiomyocytes against postischemic mitochondrial damage. Soluble epoxide hydrolase 2 (EPHX2) diminishes the benefits of EETs through hydrolysis to less active dihydroxyeicosatrienoic acids. EPHX2 inhibition or genetic disruption improves recovery of cardiac function after ischemia. Immunohistochemical staining revealed EPHX2 expression in cardiomyocytes and some endothelial cells but little expression in cardiac smooth muscle cells or fibroblasts. To determine specific roles of EPHX2 in cardiac cell types, we generated mice with cell-specific disruption of Ephx2 in endothelial cells (Ephx2fx/fx/Tek-cre) or cardiomyocytes (Ephx2fx/fx/Myh6-cre) to compare to global Ephx2-deficient mice (global Ephx2-/-) and WT (Ephx2fx/fx) mice in expression, EET hydrolase activity, and heart function studies. Most cardiac EPHX2 expression and activity is in cardiomyocytes with substantially less activity in endothelial cells. Ephx2fx/fx/Tek-cre hearts have similar EPHX2 expression, hydrolase activity, and postischemic cardiac function as control Ephx2fx/fx hearts. However, Ephx2fx/fx/Myh6-cre hearts were similar to global Ephx2-/- hearts with significantly diminished EPHX2 expression, decreased hydrolase activity, and enhanced postischemic cardiac function compared to Ephx2fx/fx hearts. During reperfusion, Ephx2fx/fx/Myh6-cre hearts displayed increased ERK activation compared to Ephx2fx/fx hearts, which could be reversed by EEZE treatment. EPHX2 did not regulate coronary vasodilation in this model. We conclude that EPHX2 is primarily expressed in cardiomyocytes where it regulates EET hydrolysis and postischemic cardiac function, whereas endothelial EPHX2 does not play a significant role in these processes.

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 >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.

iCre recombinase expressed in the anti-Müllerian hormone receptor 2 gene causes global genetic modification in the mouse†.

Genetically engineered mice are widely used to study the impact of altered gene expression in vivo. Within the reproductive tract, the Amhr2-IRES-Cre(Bhr) mouse model is used to ablate genes in ovarian granulosa and uterine stromal cells. There are reports of Amhr2-IRES-Cre(Bhr) inducing recombination in non-target tissues. We hypothesized the inefficiency or off-target Cre action in Amhr2-IRES-Cre(Bhr) mice is due to lack of recombination in every cell that expresses Amhr2. To investigate, we created a new targeted knock-in mouse model, Amhr2-iCre(Fjd), by inserting a codon-optimized improved Cre (iCre) into exon 1 of the Amhr2 gene. Amhr2-iCre(Fjd)/+ males were mated with females that contain a lox-stop-lox cassette in the Sun1 gene so when DNA recombination occurs, SUN1-sfGFP fusion protein is expressed in a peri-nuclear pattern. In adult Amhr2-iCre(Fjd)/+ Sun1LsL/+ mice, Amhr2-iCre(Fjd)-mediated genetic recombination was apparent in uterine epithelial, stromal, and myometrial cells, while Amhr2-IRES-Cre(Bhr)/+ Sun1LsL/+ females demonstrated inter-mouse variability of Amhr2-IRES-Cre(Bhr) activity in uterine cells. Fluorescence was observed in Amhr2-iCre(Fjd)-positive mice at post-natal Day 1, indicating global genetic recombination, while fluorescence of individual Amhr2-IRES-Cre(Bhr)-positive pups varied. To determine the developmental stage that genetic recombination first occurs, Sun1LsL/LsL females were super-ovulated and mated with Amhr2-IRES-Cre(Bhr)/+ or Amhr2(iCre/+)Fjd males, then putative zygotes were collected and cultured. In the four-cell embryo, Amhr2-iCre(Fjd) and Amhr2-IRES-Cre(Bhr) activities were apparent in 100% and 25-100% of cells, respectively. In conclusion, Amhr2-IRES-Cre(Bhr) or Amhr2-iCre(Fjd) driven by the Amhr2 promoter is active in the early embryo and can lead to global genetic modification, rendering this transgenic mouse model ineffective.

Inserting Cre recombinase into the Prolactin 8a2 gene for decidua-specific recombination in mice.

An estimated 75% of unsuccessful pregnancies are due to implantation failure. Investigating the causes of implantation failure is difficult as decidualization and embryo implantation is a dynamic process. Here, we describe a new decidua-specific iCre recombinase mouse strain. Utilizing CRISPR/Cas9-based genome editing, a mouse strain was developed that expresses iCre recombinase under the control of the endogenous prolactin family 8, subfamily a, member 2 (Prl8a2) promoter. iCre recombinase activity was examined by crossing with mTmG/+ or Sun1-GFP reporter alleles. iCre activity initiated reporter expression at gestational day 5.5 in the primary decidual zone and continued into mid-gestation (gestational day 9.5), with expression highly concentrated in the anti-mesometrial region. No reporter expression was observed in the ovary, oviduct, pituitary, or skeletal muscle, supporting the tissue specificity of the Prl8a2iCre in the primary decidual zone. This novel iCre line will be a valuable tool for in vivo genetic manipulation and lineage tracing to investigate functions of genetic networks and cellular dynamics associated with decidualization and infertility.

Cardiomyocyte-specific disruption of soluble epoxide hydrolase limits inflammation to preserve cardiac function.

Endotoxemia elicits a multiorgan inflammatory response that results in cardiac dysfunction and often leads to death. Inflammation-induced metabolism of endogenous N-3 and N-6 polyunsaturated fatty acids generates numerous lipid mediators, such as epoxy fatty acids (EpFAs), which protect the heart. However, EpFAs are hydrolyzed by soluble epoxide hydrolase (sEH), which attenuates their cardioprotective actions. Global genetic disruption of sEH preserves EpFA levels and attenuates cardiac dysfunction in mice following acute lipopolysaccharide (LPS)-induced inflammatory injury. In leukocytes, EpFAs modulate the innate immune system through the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. However, the mechanisms by which both EpFAs and sEH inhibition exert their protective effects in the cardiomyocyte are still elusive. This study investigated whether cardiomyocyte-specific sEH disruption attenuates inflammation and cardiac dysfunction in acute LPS inflammatory injury via modulation of the NLRP3 inflammasome. We use tamoxifen-inducible CreER recombinase technology to target sEH genetic disruption to the cardiomyocyte. Primary cardiomyocyte studies provide mechanistic insight into inflammasome signaling. For the first time, we demonstrate that cardiomyocyte-specific sEH disruption preserves cardiac function and attenuates inflammatory responses by limiting local cardiac inflammation and activation of the systemic immune response. Mechanistically, inhibition of cardiomyocyte-specific sEH activity or exogenous EpFA treatment do not prevent upregulation of NLRP3 inflammasome machinery in neonatal rat cardiomyocytes. Rather, they limit downstream activation of the pathway leading to release of fewer chemoattractant factors and recruitment of immune cells to the heart. These data emphasize that cardiomyocyte sEH is vital for mediating detrimental systemic inflammation.NEW & NOTEWORTHY The cardioprotective effects of genetic disruption and pharmacological inhibition of sEH have been demonstrated in a variety of cardiac disease models, including acute LPS inflammatory injury. For the first time, it has been demonstrated that sEH genetic disruption limited to the cardiomyocyte profoundly preserves cardiac function and limits local and systemic inflammation following acute LPS exposure. Hence, cardiomyocytes serve a critical role in the innate immune response that can be modulated to protect the heart.

Mice blocking Ser347 phosphorylation of pregnane x receptor develop hepatic fasting-induced steatosis and hypertriglyceridemia.

Nuclear receptor Pregnane X Receptor (PXR; NR1I2) has transcriptional regulation functions for energy homeostasis in the liver. Mouse PXR has a conserved phosphorylation motif at serine 347 (serine 350 in humans) within the ligand-binding domain. PXR phosphorylated at this motif is expressed in mouse livers in response to fasting. Mice with a PXR∗Ser347Ala knockin mutation (PXR KI) were generated to block phosphorylation, and utilized to investigate the role of Ser347 phosphorylation in vivo. PXR KI mice had decreased body weight at 8-weeks of age and had much greater weight loss after fasting compared with PXR WT mice. The cDNA microarray analysis of hepatic mRNAs showed that cell death or apoptotic signaling was induced in fasting PXR KI mice. Moreover, increasing hepatic lipids, triglycerides and the development of hypertriglyceridemia were observed in fasting PXR KI mice. These findings are indicative that blocking phosphorylation prevents mice from maintaining hepatic energy homeostasis. Thus, phosphorylated PXR may be an essential factor to prevent the liver from developing damage caused by fasting.

Notch effector recombination signal binding protein for immunoglobulin kappa J signaling is required for the initiation of endometrial stromal cell decidualization†.

The Notch signaling pathway is required for reproductive success. This pathway activates its transcriptional effector, recombination signal binding protein for immunoglobulin kappa J (Rbpj), to induce transcription of its target genes. This signaling pathway is required for successful decidualization, implantation, and uterine repair following parturition. To identify the compartmental specific roles of the Notch signaling pathway in the establishment of pregnancy, we generated epithelial and decidual stromal cell specific knockouts of Rbpj utilizing lactoferrin iCre and Prl8A2 iCre, respectively. Both conditional knockout mouse models were fertile. The Rbpj epithelial knockout mice displayed 27% resorption sites at E15.5, but this did not significantly impact the number of live born pups compared with controls. In addition, the Rbpj epithelial knockout mice displayed increased estrogen signaling in their stromal compartment. Given that both mouse models exhibited fertility comparable to control animals, the epithelial and stromal specific nature of the iCre recombinases utilized, and previously published Rbpj total uterine knockout mouse models, we conclude that Notch effector Rbpj signaling is required at the initiation of pregnancy to support decidualization in stromal cells, but that Rbpj is not required in the epithelial compartment nor is it required for post-implantation pregnancy success.

Noncanonical autophagy in dermal dendritic cells mediates immunosuppressive effects of UV exposure.

BACKGROUND: Control of the inflammatory response is critical to maintaining homeostasis, and failure to do so contributes to the burden of chronic inflammation associated with several disease states. The mechanisms that underlie immunosuppression, however, remain largely unknown. Although defects in autophagy machinery have been associated with inflammatory pathologic conditions, we now appreciate that autophagic components participate in noncanonical pathways distinct from classical autophagy. We have previously demonstrated that LC3-associated phagocytosis (LAP), a noncanonical autophagic process dependent on Rubicon (rubicon autophagy regulator [RUBCN]), contributes to immunosuppression. OBJECTIVE: We used Rubcn-/- mice to examine the role of the LAP pathway in mediating the UV-induced immunotolerant program in a model of contact hypersensitivity (CHS). METHODS: Flow cytometry and transcriptional analysis were used to measure immune cell infiltration and activation in the skin of Rubcn+/+ and Rubcn-/- mice during the CHS response. RESULTS: Here, we demonstrate that LAP is required for UV-induced immunosuppression and that UV exposure induces a broadly anti-inflammatory transcriptional program dependent on Rubicon. Rubcn-/- mice are resistant to UV-induced immunosuppression and instead display exaggerated inflammation in a model of CHS. Specifically, RUBCN deficiency in CD301b+ dermal dendritic cells results in their increased antigen presentation capacity and subsequent hyperactivation of the CD8+ T-cell response. CONCLUSIONS: LAP functions to limit the immune response and is critical in maintaining the balance between homeostasis and inflammation.

A distal super enhancer mediates estrogen-dependent mouse uterine-specific gene transcription of Igf1 (insulin-like growth factor 1).

Insulin-like growth factor 1 (IGF1) is primarily synthesized in and secreted from the liver; however, estrogen (E2), through E2 receptor α (ERα), increases uterine Igf1 mRNA levels. Previous ChIP-seq analyses of the murine uterus have revealed a potential enhancer region distal from the Igf1 transcription start site (TSS) with multiple E2-dependent ERα-binding regions. Here, we show E2-dependent super enhancer-associated characteristics and suggest contact between the distal enhancer and the Igf1 TSS. We hypothesized that this distal super-enhancer region controls E2-responsive induction of uterine Igf1 transcripts. We deleted 430 bp, encompassing one of the ERα-binding sites, thereby disrupting interactions of the enhancer with gene-regulatory factors. As a result, E2-mediated induction of mouse uterine Igf1 mRNA is completely eliminated, whereas hepatic Igf1 expression remains unaffected. This highlights the central role of a distal enhancer in the assembly of the factors necessary for E2-dependent interaction with the Igf1 TSS and induction of uterus-specific Igf1 transcription. Of note, loss of the enhancer did not affect fertility or uterine growth responses. Deletion of uterine Igf1 in a PgrCre;Igf1f/f model decreased female fertility but did not impact the E2-induced uterine growth response. Moreover, E2-dependent activation of uterine IGF1 signaling was not impaired by disrupting the distal enhancer or by deleting the coding transcript. This indicated a role for systemic IGF1, suggested that other growth mediators drive uterine response to E2, and suggested that uterine-derived IGF1 is essential for reproductive success. Our findings elucidate the role of a super enhancer in Igf1 regulation and uterine growth.

Leucine-rich repeats and calponin homology containing 4 (Lrch4) regulates the innate immune response.

Toll-like receptors (TLRs) are pathogen-recognition receptors that trigger the innate immune response. Recent reports have identified accessory proteins that provide essential support to TLR function through ligand delivery and receptor trafficking. Herein, we introduce leucine-rich repeats (LRRs) and calponin homology containing 4 (Lrch4) as a novel TLR accessory protein. Lrch4 is a membrane protein with nine LRRs in its predicted ectodomain. It is widely expressed across murine tissues and has two expression variants that are both regulated by lipopolysaccharide (LPS). Predictive modeling indicates that Lrch4 LRRs conform to the horseshoe-shaped structure typical of LRRs in pathogen-recognition receptors and that the best structural match in the protein database is to the variable lymphocyte receptor of the jawless vertebrate hagfish. Silencing Lrch4 attenuates cytokine induction by LPS and multiple other TLR ligands and dampens the in vivo innate immune response. Lrch4 promotes proper docking of LPS in lipid raft membrane microdomains. We provide evidence that this is through regulation of lipid rafts as Lrch4 silencing reduces cell surface gangliosides, a metric of raft abundance, as well as expression and surface display of CD14, a raft-resident LPS co-receptor. Taken together, we identify Lrch4 as a broad-spanning regulator of the innate immune response and a potential molecular target in inflammatory disease.

Genome-wide identification of FOXL2 binding and characterization of FOXL2 feminizing action in the fetal gonads.

The identity of the gonads is determined by which fate, ovarian granulosa cell or testicular Sertoli cell, the bipotential somatic cell precursors choose to follow. In most vertebrates, the conserved transcription factor FOXL2 contributes to the fate of granulosa cells. To understand FOXL2 functions during gonad differentiation, we performed genome-wide analysis of FOXL2 chromatin occupancy in fetal ovaries and established a genetic mouse model that forces Foxl2 expression in the fetal testis. When FOXL2 was ectopically expressed in the somatic cell precursors in the fetal testis, FOXL2 was sufficient to repress Sertoli cell differentiation, ultimately resulting in partial testis-to-ovary sex-reversal. Combining genome-wide analysis of FOXL2 binding in the fetal ovary with transcriptomic analyses of our Foxl2 gain-of-function and previously published Foxl2 loss-of-function models, we identified potential pathways responsible for the feminizing action of FOXL2. Finally, comparison of FOXL2 genome-wide occupancy in the fetal ovary with testis-determining factor SOX9 genome-wide occupancy in the fetal testis revealed extensive overlaps, implying that antagonistic signals between FOXL2 and SOX9 occur at the chromatin level.

Transethnic associations among immune-mediated diseases and single-nucleotide polymorphisms of the aryl hydrocarbon response gene ARNT and the PTPN22 immune regulatory gene.

BACKGROUND: Because immune responses are sensitive to environmental changes that drive selection of genetic variants, we hypothesized that polymorphisms of some xenobiotic response and immune response genes may be associated with specific types of immune-mediated diseases (IMD), while others may be associated with IMD as a larger category regardless of specific phenotype or ethnicity. OBJECTIVE: To examine transethnic gene-IMD associations for single nucleotide polymorphism (SNP) frequencies of prototypic xenobiotic response genes-aryl hydrocarbon receptor (AHR), AHR nuclear translocator (ARNT), AHR repressor (AHRR) - and a prototypic immune response gene, protein tyrosine phosphatase, non-receptor type 22 (PTPN22), in subjects from the Environmental Polymorphisms Registry (EPR). METHODS: Subjects (n = 3731) were genotyped for 14 SNPs associated with functional variants of the AHR, ARNT, AHRR, and PTPN22 genes, and their frequencies were compared among African Americans (n = 1562), Caucasians (n = 1838), and Hispanics (n = 331) with previously reported data. Of those genotyped, 2015 EPR subjects completed a Health and Exposure survey. SNPs were assessed via PLINK for associations with IMD, which included those with autoimmune diseases, allergic disorders, asthma, or idiopathic pulmonary fibrosis. Transethnic meta-analyses were performed using METAL and MANTRA approaches. RESULTS: ARNT SNP rs11204735 was significantly associated with autoimmune disease by transethnic meta-analyses using METAL (odds ratio, OR [95% confidence interval] = 1.29 [1.08-1.55]) and MANTRA (ORs ranged from 1.29 to 1.30), whereas ARNT SNP rs1889740 showed a significant association with autoimmune disease by METAL (OR = 1.25 [1.06-1.47]). For Caucasian females, PTPN22 SNP rs2476601 was significantly associated with autoimmune disease by allelic association tests (OR = 1.99, [1.30-3.04]). In Caucasians and Caucasian males, PTPN22 SNP rs3811021 was significantly associated with IMD (OR = 1.39 [1.12-1.72] and 1.50 [1.12-2.02], respectively) and allergic disease (OR = 1.39 [1.12-1.71], and 1.62 [1.19-2.20], respectively). In the transethnic meta-analysis, PTPN22 SNP rs3811021 was significantly implicated in IMD by METAL (OR = 1.31 [1.10-1.56]), and both METAL and MANTRA suggested that rs3811021 was associated with IMD and allergic disease in males across all three ethnic groups (IMD METAL OR = 1.50 [1.15-1.95]; IMD MANTRA ORs ranged from 1.47 to 1.50; allergic disease METAL OR = 1.58 [1.20-2.08]; allergic disease MANTRA ORs ranged from 1.55 to 1.59). CONCLUSIONS: Some xenobiotic and immune response gene polymorphisms were shown here, for the first time, to have associations across a broad spectrum of IMD and ethnicities. Our findings also suggest a role for ARNT in the development of autoimmune diseases, implicating environmental factors metabolized by this pathway in pathogenesis. Further studies are needed to confirm these data, assess the implications of these findings, define gene-environment interactions, and explore the mechanisms leading to these increasingly prevalent disorders.

Expression of Cyp2c/Cyp2j subfamily members and oxylipin levels during LPS-induced inflammation and resolution in mice.

Inflammatory stimuli, such as bacterial LPS, alter the expression of many cytochromes P450. CYP2C and CYP2J subfamily members actively metabolize fatty acids to bioactive eicosanoids, which exhibit potent anti-inflammatory effects. Herein, we examined mRNA levels of the 15 mouse Cyp2c and 7 mouse Cyp2j isoforms in liver, kidney, duodenum, and brain over a 96-h time course of LPS-induced inflammation and resolution. Plasma and liver eicosanoid levels were also measured by liquid chromatography with tandem mass spectrometry. Expression changes in Cyp2c and Cyp2j isoforms were both isoform and tissue specific. Total liver Cyp2c and Cyp2j mRNA content was reduced by 80% 24 h after LPS but recovered to baseline levels by 96 h. Total Cyp2c and Cyp2j mRNA in kidney (-19%) and duodenum (-64%) were reduced 24 h after LPS but recovered above baseline by 72 h. Total Cyp2c and Cyp2j mRNA content in brain was elevated at all time points after LPS dosing. Plasma eicosanoids transiently increased 3-6 h after administration of LPS. In liver, esterified oxylipin levels decreased during acute inflammation and before recovering. The biphasic suppression and recovery of mouse Cyp2c and Cyp2j isoforms and associated changes in eicosanoid levels during LPS-induced inflammation and resolution may have important physiologic consequences.-Graves, J. P., Bradbury, J. A., Gruzdev, A., Li, H., Duval, C., Lih, F. B., Edin, M. L., Zeldin, D. C. Expression of Cyp2c/Cyp2j subfamily members and oxylipin levels during LPS-induced inflammation and resolution in mice.

Epoxide hydrolase 1 (EPHX1) hydrolyzes epoxyeicosanoids and impairs cardiac recovery after ischemia.

Stimuli such as inflammation or hypoxia induce cytochrome P450 epoxygenase-mediated production of arachidonic acid-derived epoxyeicosatrienoic acids (EETs). EETs have cardioprotective, vasodilatory, angiogenic, anti-inflammatory, and analgesic effects, which are diminished by EET hydrolysis yielding biologically less active dihydroxyeicosatrienoic acids (DHETs). Previous in vitro assays have suggested that epoxide hydrolase 2 (EPHX2) is responsible for nearly all EET hydrolysis. EPHX1, which exhibits slow EET hydrolysis in vitro, is thought to contribute only marginally to EET hydrolysis. Using Ephx1-/-, Ephx2-/-, and Ephx1-/-Ephx2-/- mice, we show here that EPHX1 significantly contributes to EET hydrolysis in vivo Disruption of Ephx1 and/or Ephx2 genes did not induce compensatory changes in expression of other Ephx genes or CYP2 family epoxygenases. Plasma levels of 8,9-, 11,12-, and 14,15-DHET were reduced by 38, 44, and 67% in Ephx2-/- mice compared with wildtype (WT) mice, respectively; however, plasma from Ephx1-/-Ephx2-/- mice exhibited significantly greater reduction (100, 99, and 96%) of those respective DHETs. Kinetic assays and FRET experiments indicated that EPHX1 is a slow EET scavenger, but hydrolyzes EETs in a coupled reaction with cytochrome P450 to limit basal EET levels. Moreover, we also found that EPHX1 activities are biologically relevant, as Ephx1-/-Ephx2-/- hearts had significantly better postischemic functional recovery (71%) than both WT (31%) and Ephx2-/- (51%) hearts. These findings indicate that Ephx1-/-Ephx2-/- mice are a valuable model for assessing EET-mediated effects, uncover a new paradigm for EET metabolism, and suggest that dual EPHX1 and EPHX2 inhibition may represent a therapeutic approach to manage human pathologies such as myocardial infarction.

20-HETE Signals Through G-Protein-Coupled Receptor GPR75 (Gq) to Affect Vascular Function and Trigger Hypertension.

RATIONALE: 20-Hydroxyeicosatetraenoic acid (20-HETE), one of the principle cytochrome P450 eicosanoids, is a potent vasoactive lipid whose vascular effects include stimulation of smooth muscle contractility, migration, and proliferation, as well as endothelial cell dysfunction and inflammation. Increased levels of 20-HETE in experimental animals and in humans are associated with hypertension, stroke, myocardial infarction, and vascular diseases. OBJECTIVE: To date, a receptor/binding site for 20-HETE has been implicated based on the use of specific agonists and antagonists. The present study was undertaken to identify a receptor to which 20-HETE binds and through which it activates a signaling cascade that culminates in many of the functional outcomes attributed to 20-HETE in vitro and in vivo. METHODS AND RESULTS: Using crosslinking analogs, click chemistry, binding assays, and functional assays, we identified G-protein receptor 75 (GPR75), currently an orphan G-protein-coupled receptor (GPCR), as a specific target of 20-HETE. In cultured human endothelial cells, 20-HETE binding to GPR75 stimulated Gαq/11 protein dissociation and increased inositol phosphate accumulation and GPCR-kinase interacting protein-1-GPR75 binding, which further facilitated the c-Src-mediated transactivation of epidermal growth factor receptor. This results in downstream signaling pathways that induce angiotensin-converting enzyme expression and endothelial dysfunction. Knockdown of GPR75 or GPCR-kinase interacting protein-1 prevented 20-HETE-mediated endothelial growth factor receptor phosphorylation and angiotensin-converting enzyme induction. In vascular smooth muscle cells, GPR75-20-HETE pairing is associated with Gαq/11- and GPCR-kinase interacting protein-1-mediated protein kinase C-stimulated phosphorylation of MaxiKß, linking GPR75 activation to 20-HETE-mediated vasoconstriction. GPR75 knockdown in a mouse model of 20-HETE-dependent hypertension prevented blood pressure elevation and 20-HETE-mediated increases in angiotensin-converting enzyme expression, endothelial dysfunction, smooth muscle contractility, and vascular remodeling. CONCLUSIONS: This is the first report to identify a GPCR target for an eicosanoid of this class. The discovery of 20-HETE-GPR75 pairing presented here provides the molecular basis for the signaling and pathophysiological functions mediated by 20-HETE in hypertension and cardiovascular diseases.

En masse lentiviral gene delivery to mouse fertilized eggs via laser perforation of zona pellucida.

Lentiviruses are highly efficient vehicles for delivering genes into cells. They readily transduce primary and immortalized cells in vivo and in vitro. Genes delivered by lentiviruses are incorporated and replicated as part of their host genome and therefore offer a powerful tool for creation of stable cell lines and transgenic animals. However, the zona pellucida surrounding the fertilized eggs acts as a barrier and hinders lentiviral transduction of embryos. Here, we utilize a laser, typically used to perforate the zona pellucida for in vitro fertilization, to permeabilize the zona for lentiviral gene delivery. A single hole in the zona is sufficient for the lentivirus to gain access to fertilized eggs without the need for microinjection for en masse gene delivery. Embryos generated by this method elicit no damage and can develop to term for creation of transgenic animals.

Characterization of the Tissue Distribution of the Mouse Cyp2c Subfamily by Quantitative PCR Analysis.

The CYP2C subfamily of the cytochrome P450 gene superfamily encodes heme-thiolate proteins that have a myriad of biologic functions. CYP2C proteins detoxify xenobiotics and metabolize endogenous lipids such as arachidonic acid to bioactive eicosanoids. We report new methods and results for the quantitative polymerase reaction (qPCR) analysis for the 15 members of the mouse Cyp2c subfamily (Cyp2c29, Cyp2c37, Cyp2c38, Cyp2c39, Cyp2c40, Cyp2c44, Cyp2c50, Cyp2c54, Cyp2c55, Cyp2c65, Cyp2c66, Cyp2c67, Cyp2c68, Cyp2c69, and Cyp2c70). Commercially available TaqMan primer/probe assays were compared with developed SYBR Green primer sets for specificity toward the mouse Cyp2c cDNAs and analysis of their tissue distribution. TaqMan primer/probe assays for 10 of the mouse Cyp2c isoforms were shown to be specific for their intended mouse Cyp2c cDNA; however, there were no TaqMan primer/probe assays specific for the mouse Cyp2c29, Cyp2c40, Cyp2c67, Cyp2c68, or Cyp2c69 transcripts. Each of the SYBR Green primer sets was specific for its intended mouse Cyp2c cDNA. The two qPCR methods confirmed similar patterns of Cyp2c tissue expression: Cyp2c37, Cyp2c38, Cyp2c39, Cyp2c44, Cyp2c50, Cyp2c54, and Cyp2c70 were most highly expressed in liver; Cyp2c55 was highly expressed in large intestine; Cyp2c65 was highly expressed in stomach, duodenum, and large intestine; and Cyp2c66 was highly expressed in both duodenum and jejunum. For isoforms without specific TaqMan primer/probe assays, the SYBR Green primer sets detected high level expression of Cyp2c29, Cyp2c40, Cyp2c67, Cyp2c68, and Cyp2c69 in the liver. Lower expression levels of the mouse Cyp2cs were also detected in other tissues.

Contribution of alveolar type II cell-derived cyclooxygenase-2 to basal airway function, lung inflammation, and lung fibrosis.

Cyclooxygenase (COX)-2 has been shown to be involved in regulating basal airway function, bacterial LPS-induced airway hyperresponsiveness (AHR) and lung inflammation, and bleomycin-induced lung fibrosis; however, the cellular source of COX-2 that underlies these effects is unknown. We generated mice with alveolar type II (ATII) cell-specific knockdown of COX-2 (AT2CC(-/-)), to examine the role of ATII cell-derived prostaglandins (PGs) in these processes. Specific knockdown of COX-2 was confirmed by real-time RT-PCR and Western blot analyses. LC/MS/MS analysis showed that ATII cells produced PGs. Basal airway responsiveness of AT2CC(-/-) mice was decreased compared to that of wild-type (WT) mice. LPS-induced hypothermic response, infiltration of inflammatory cells into the airway, and lung inflammation were enhanced in AT2CC(-/-) mice relative to WT controls; however, LPS-induced AHR and proinflammatory cytokine and chemokine expression were similar between the genotypes. After 21 d of bleomycin administration, AT2CC(-/-) mice behaved in a manner similar to WT mice. Thus, ATII cell-derived COX-2 plays an important role in regulating basal airway function and LPS-induced lung inflammation, but does not play a role in bleomycin-induced fibrosis. These findings provide insight into the cellular source of COX-2 related to these lung phenotypes.