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...