ABSTRACT:Cytochrome P450 (P450) eicosanoids regulate vascular tone, renal tubular transport, cellular proliferation, and inflammation. Both the CYP4A -hydroxylases, which catalyze 20-hydroxyeicosatetraenoic acid (20-HETE) formation, and soluble epoxide hydrolase (sEH), which catalyzes epoxyeicosatrienoic acid (EET) degradation to the dihydroxyeicosatrienoic acids (DHETs), are induced upon activation of peroxisome proliferator-activated receptor ␣ (PPAR␣) by fatty acids and fibrates. In contrast, the CYP2C epoxygenases, which are responsible for EET formation, are repressed after fibrate treatment. We show here that P450 eicosanoids can bind to and activate PPAR␣ and result in the modulation of PPAR␣ target gene expression. In transactivation assays, 14,15-DHET, 11,2-EET, and 20-HETE were potent activators of PPAR␣. Gel shift assays showed that EETs, DHETs, and 20-HETE induced PPAR␣-specific binding to its cognate response element. Expression of apolipoprotein A-I was decreased 70% by 20-HETE, whereas apolipoprotein A-II expression was increased up to 3-fold by 11,12-EET, 14,15-DHET, and 20-HETE. In addition, P450 eicosanoids induced CYP4A1, sEH, and CYP2C11 expression, suggesting that they can regulate their own levels. Given that P450 eicosanoids have multiple cardiovascular effects, pharmacological modulation of their formation and/or degradation may yield therapeutic benefits.

Human embryonic stem cells (hESCs) have the capacity to remain pluripotent and self-renew indefinitely. To discover novel players in the maintenance of hESCs, we have previously reported the generation of monoclonal antibodies that bind to cell surface markers on hESCs, and not to mouse embryonic stem cells or differentiated embryoid bodies. In this study, we have identified the antigen target of one such monoclonal antibody as the epithelial cell adhesion molecule (EpCAM). In undifferentiated hESCs, EpCAM is localized to Octamer 4 (OCT4)-positive pluripotent cells, and its expression is down-regulated upon differentiation. To further understand its biological function in hESCs, endogenous EpCAM expression was silenced using small interfering RNA. EpCAM knockdown had marginal negative effects on OCT4 and TRA-1-60 expression, however cell proliferation was decreased by >40%.Examination of lineage marker expression showed marked upregulation of endoderm and mesoderm genes in EpCAM-silenced cells, under both pluripotent and differentiating conditions. These results were validated using a hESC line whose EpCAM expression has been stably knocked down. Data from the stable line confirmed that downregulation of EpCAM decreases cell growth and increases gene expression in the endoderm and mesoderm lineages. In vivo, hESCs lacking EpCAM were able to form teratomas containing tissues representing the three germ layers, and gene expression analysis yielded marked increase in the endoderm marker alpha fetoprotein compared with control. Together these data demonstrate that EpCAM is a surface marker on undifferentiated hESCs and plays functional roles in proliferation and differentiation. STEM CELLS 2010;28:29-35 Disclosure of potential conflicts of interest is found at the end of this article.

The role of cyclo‐oxygenase (COX) in the regulation of anion secretion (measured as short‐ circuit current, Isc) in cultured epididymal epithelia from immature rats was investigated. COX inhibitors attenuated the increase of anion secretion caused by bradykinin (LBK) but had no effect on that caused by PGE2, suggesting that prostaglandin synthesis mediates the secretory response of the tissues to LBK. The apparent IC50 values for indomethacin, piroxicam and L‐745,337 in inhibiting the LBK‐induced Isc were 0·14, 1·34 and 15·7 μM, respectively. This order of potency: indomethacin > piroxicam > L‐745,337 >> DFU suggests the involvement of the COX‐1 isozyme in the mediation of the secretory response to LBK. Among the COX products (prostaglandins, thromboxane and prostacyclins) tested, only PGE2 and, to a much lesser extent, PGF2α stimulated anion secretion by cultured rat epididymal epithelia. The effect of PGE2 was mimicked by 11‐deoxyl PGE1, a specific prostaglandin E (EP)2/4 receptor agonist, but not by sulprostone, a specific EP1/3 receptor agonist, indicating that cyclic AMP‐coupled EP2/4 receptors are involved in the LBK‐stimulated anion secretion. A reverse transcriptase‐polymerase chain reaction study detected the expression of COX‐1 and COX‐2 mRNA in intact rat epididymis and in cultured epididymal epithelia. The expression of COX‐1 mRNA was reduced by LBK by 44 %. Immunohistochemical studies demonstrated the presence of COX‐1 immunoreactivity in the basal cells of the intact rat epididymis. By comparision, COX‐2 immunoreactivity was detected in the apical pole of the principal cells. The role of COX in the formation of the epididymal microenvironment and the implication of long term administration of non‐steroidal anti‐inflammatory drugs (NSAIDs) on male fertility are discussed.

Objective-Proliferation of smooth muscle cells is implicated in cardiovascular complications. Previously, a urea-based soluble epoxide hydrolase inhibitor was shown to attenuate smooth muscle cell proliferation. We examined the possibility that urea-based alkanoic acids activate the nuclear receptor peroxisome proliferator-activated receptor ␣ (PPAR␣) and the role of PPAR␣ in smooth muscle cell proliferation. Methods and Results-Alkanoic acids transactivated PPAR␣, induced binding of PPAR␣ to its response element, and significantly induced the expression of PPAR␣-responsive genes, showing their function as PPAR␣ agonists. Furthermore, the alkanoic acids attenuated platelet-derived growth factor-induced smooth muscle cell proliferation via repression of cyclin D1 expression. Using small interfering RNA to decrease endogenous PPAR␣ expression, it was determined that PPAR␣ was partially involved in the cyclin D1 repression. The antiproliferative effects of alkanoic acids may also be attributed to their inhibitory effects on soluble epoxide hydrolase, because epoxyeicosatrienoic acids alone inhibited smooth muscle cell proliferation. Conclusions-These results show that attenuation of smooth muscle cell proliferation by urea-based alkanoic acids is mediated, in part, by the activation of PPAR␣. These acids may be useful for designing therapeutics to treat diseases characterized by excessive smooth muscle cell proliferation. Key Words: soluble expoxide hydrolase Ⅲ epoxyeicosatrienoic acids Ⅲ PPAR␣ Ⅲ smooth muscle cells Ⅲ proliferation S mooth muscle cell (SMC) proliferation is a critical event in atherosclerosis 1 and in restenosis following interventional procedures. 2 After injury to the vasculature, a vasculoproliferative cascade is initiated that includes the recruitment and proliferation of SMCs, 3 which can eventually lead to occlusive lesions that result in myocardial ischemia. 4 Much effort has been made to inhibit SMC proliferation using pharmacological and genetic approaches that interfere with cell cycle regulators such as cyclins 5 and cyclin-dependent kinases (CDKs). 6 Cyclins and CDKs are part of the regulatory machinery controlling cell cycle progression. The D and E cyclins and their associated kinases are viewed as essential for entry into and progression through the G 1 phase of a cell cycle. 7 Overexpression of the D cyclins can shorten G 1 , implying that they are critical for cell cycle progression through this phase. 8 In leukemic and breast cancer cells, xenobiotics such as clofibrate and troglitazone inhibit cyclin D expression, resulting in cell cycle arrest. 9,10 These compounds are ligands for the peroxisome proliferator-activated receptor (PPAR) (NR1C) family of nuclear receptors.PPARs are ligand-activated nuclear receptors of which there are 3 isoforms (␣, ␥, and ␦). Activators of PPAR␣ include polyunsaturated fatty acids and fibrate drugs. PPAR␥ ligands include the prostaglandin (PG) D 2 derivative 15-deoxy-⌬ 12,14 -prostaglindin J 2 (15-⌬PGJ 2 ), oxidized linoleic acid, and the antidiabe...

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ImportanceResearch evidence is mounting for the association between infant screen use and negative cognitive outcomes related to attention and executive functions. The nature, timing, and persistence of screen time exposure on neural functions are currently unknown. Electroencephalography (EEG) permits elucidation of the neural correlates associated with cognitive impairments.ObjectiveTo examine the associations between infant screen time, EEG markers, and school-age cognitive outcomes using mediation analysis with structural equation modeling.Design, Setting, and ParticipantsThis prospective maternal-child dyad cohort study included participants from the population-based study Growing Up in Singapore Toward Healthy Outcomes (GUSTO). Pregnant mothers were enrolled in their first trimester from June 2009 through December 2010. A subset of children who completed neurodevelopmental visits at ages 12 months and 9 years had EEG performed at age 18 months. Data were reported from 3 time points at ages 12 months, 18 months, and 9 years. Mediation analyses were used to investigate how neural correlates were involved in the paths from infant screen time to the latent construct of attention and executive functioning. Data for this study were collected from November 2010 to March 2020 and were analyzed between October 2021 and May 2022.ExposuresParent-reported screen time at age 12 months.Main Outcomes and MeasuresPower spectral density from EEG was collected at age 18 months. Child attention and executive functions were measured with teacher-reported questionnaires and objective laboratory-based tasks at age 9 years.ResultsIn this sample of 437 children, the mean (SD) age at follow-up was 8.84 (0.07) years, and 227 children (51.9%) were male. The mean (SD) amount of daily screen time at age 12 months was 2.01 (1.86) hours. Screen time at age 12 months contributed to multiple 9-year attention and executive functioning measures (η2, 0.03-0.16; Cohen d, 0.35-0.87). A subset of 157 children had EEG performed at age 18 months; EEG relative theta power and theta/beta ratio at the frontocentral and parietal regions showed a graded correlation with 12-month screen use (r = 0.35-0.37). In the structural equation model accounting for household income, frontocentral and parietal theta/beta ratios partially mediated the association between infant screen time and executive functioning at school age (exposure-mediator β, 0.41; 95% CI, 0.22 to 0.59; mediator-outcome β, −0.38; 95% CI, −0.64 to −0.11), forming an indirect path that accounted for 39.4% of the association.Conclusions and RelevanceIn this study, infant screen use was associated with altered cortical EEG activity before age 2 years; the identified EEG markers mediated the association between infant screen time and executive functions. Further efforts are urgently needed to distinguish the direct association of infant screen use compared with family factors that predispose early screen use on executive function impairments.

The saturated C20 isoprenoid phytanic acid is physiologically derived from phytol released in the degradation of chlorophyll. The presence of a C-3 methyl group in this substrate blocks normal ␤-oxidation, so phytanic acid degradation primarily occurs by initial peroxisomal ␣-oxidation to shift the register of the methyl group. However, individuals with Refsum's disease are genetically deficient in the required phytanoyl-CoA ␣-hydroxylase and suffer from neurological pathologies caused by the accumulation of phytanic acid. Recent work has shown that phytanic acid can also be catabolized by a pathway initiated by -hydroxylation of the hydrocarbon chain, followed by oxidation of the alcohol to the acid and conventional ␤-oxidation. However, the enzymes responsible for the -hydroxylation of phytanic acid have not been identified. In this study, we have determined the activities of all of the rat and human CYP4A enzymes and two of the rat CYP4F enzymes, with respect to the -hydroxylation of phytanic acid. Furthermore, we have shown that the ability to -hydroxylate phytanic acid is elevated in microsomes from rats pretreated with clofibrate. The results support a possible role for CYP4 enzyme elevation in the elimination of phytanic acid in Refsum's disease patients.

Dietary ␥-linolenic acid (GLA), a -6 polyunsaturated fatty acid found in borage oil (BOR), lowers systolic blood pressure in spontaneously hypertensive rats (SHRs). GLA is converted into arachidonic acid (AA) by elongation and desaturation steps. Epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) are cytochrome P450 (P450)-derived AA eicosanoids with important roles in regulating blood pressure. This study tested the hypothesis that the blood pressure-lowering effect of a GLA-enriched diet involves alteration of P450-catalyzed AA metabolism. Microsomes and RNA were isolated from the renal cortex of male SHRs fed a basal fat-free diet for 5 weeks to which 11% by weight of sesame oil (SES) or BOR was added. There was a 2.6-to 3.5-fold increase in P450 epoxygenase activity in renal microsomes isolated from the BOR-fed SHRs compared with the SES-fed rats. Epoxygenase activity accounted for 58% of the total AA metabolism in the BOR-treated kidney microsomes compared with 33% in the SES-treated rats. More importantly, renal 14,15-and 8,9-EET levels increased 1.6-to 2.5-fold after dietary BOR treatment. The increase in EET formation is consistent with increases in CYP2C23, CYP2C11, and CYP2J protein levels. There were no differences in the level of renal P450 epoxygenase mRNA between the SES-and BOR-treated rats. Enhanced synthesis of the vasodilatory EETs and decreased formation of the vasoconstrictive 20-HETE suggests that changes in P450-mediated AA metabolism may contribute, at least in part, to the blood pressure-lowering effect of a BOR-enriched diet.