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Fatty acids are an essential component of mammalian bodies. They go through different metabolic pathways depending on physiological states and inflammatory stimuli. In this study, we conducted a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based comprehensive analysis of lipid metabolites in urine of canine patients with liver mass. There were significant differences in quantity of some lipid metabolites that may be closely associated with the disease and/or general inflammatory responses, including increased metabolites of prostaglandin E

Polyunsaturated fatty acids (PUFAs), including arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), are metabolized to various lipid mediators. The profile of these lipid metabolites excreted into the urine reflects inflammatory state of the body and disease conditions. In this study, we quantified 156 types of lipids in urine samples of dogs with splenic mass, using liquid chromatography-tandem mass spectrometry. We found that metabolites of prostaglandin (PG) E

Polyunsaturated fatty acids, including arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), are converted to hundreds of lipid mediators by cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 (CYP), or through non-enzymatic processes, and they reflect inflammatory states of the body. We comprehensively analyzed lipid metabolites in dog urine using a liquid chromatograph-mass spectrometry (LC-MS/MS) to describe their metabolic characteristics. We detected 31 AA-derived metabolites, four EPA-derived metabolites, and a DHA-derived metabolite in all urine samples. Among AA-derived metabolites, 15, 5, 3, and 8 were generated by COX, LOX, CYP, and non-enzymatic oxidation respectively. This study will be the first step to use profiles of urinary lipid metabolites for better understanding and diagnosis of canine diseases.

Background and Purpose PGE2 is a major prostanoid that regulates inflammation by stimulating EP1–4 receptors. However, how PGE2 induces an initial inflammatory response to vascular hyper-permeability remains unknown. Here we investigated the role of the PGE2-EP receptor signal in modulating vascular permeability both in vivo and in vitro. Experimental Approach We used a modified Miles assay and intravital microscopy to examine vascular permeability in vivo. Endothelial barrier property was assessed by measuring transendothelial electrical resistance (TER) in vitro. Key Results Local administration of PGE2, an EP2 or EP4 receptor agonist into FVB/NJcl mouse ear skin caused vascular leakage, indicated by dye extravasation. Intravital microscopy and laser Doppler blood-flow imaging revealed that these treatments dilated peripheral vessels and increased local blood flow. Pretreatment with the vasoconstrictor phenylephrine inhibited the PGE2-induced blood flow increase and vascular leakage. In contrast to the EP2 and EP4 receptor agonists, administration of an EP3 receptor agonist suppressed vascular leakage without altering vascular diameter or blood flow. In isolated HUVECs, the EP3 receptor agonist elevated TER and blocked thrombin-induced dextran passage. Inhibiting PKA restored the hypo-permeability induced by the EP3 receptor agonist. Conclusions and Implications Activation of the PGE2-EP2 or -EP4 receptor signal induces vasodilatation in mural cells, resulting in increased local blood flow and hyper-permeability. In contrast, activation of the PGE2-EP3 receptor signal induces a cAMP-dependent enhancement of the endothelial barrier, leading to hypo-permeability. We provide the first evidence that endothelial cells and mural cells cooperate to modulate vascular permeability.

Prostaglandin E2 (PGE2) is a major prostanoid produced under inflammatory situations. There have been controversial reports showing contractile or relaxant effect of PGE2 on vascular tone in various types of blood vessels. Thus, it is still elusive whether and how PGE2 modulates vascular tone. We here assessed the effects of PGE2 on vascular contractility using different types of vasculatures isolated form rat. In endothelium-denuded aortas and mesenteric arteries, PGE2 (1 nM–10 μM) concentration-dependently enhanced the contraction elicited by K+ (35.4 mM) or norepinephrine (10 nM). In pulmonary arteries, PGE2 did not alter the both-induced contraction. Tail arteries were relaxed by a low dose of PGE2 (1–100 nM), but this response shifted to contraction by the higher dose of PGE2 (300 nM–10 μM). There are four types of PGE2 receptors EP1-4. RT-PCR showed that aortas and mesenteric arteries abundantly expressed EP3, while tail arteries abundantly expressed EP4. We next revealed that selective EP3 agonism enhanced the contraction in mesenteric arteries, whereas EP4 agonism induced relaxation in tail arteries. Taken together, PGE2 causes different contractile responses depending on the type of vascular bed. This phenomenon may be due to the difference in expression pattern and activity of EP receptors.

Lung fibroblasts are responsible for collagen secretion during normal tissue repair and the development of fibrosis. Many other prostaglandins have been reported to regulate collagen synthesis in lung fibroblasts, but the role of prostaglandin D2 (PGD2) is unknown. In this study, we investigated the effect of PGD2 on type I collagen secretion in human lung fibroblasts. Pretreatment with PGD2 (0.1 – 10 μM, 1 h) significantly attenuated type I collagen secretion to the cell supernatant induced by transforming growth factor-β (TGF-β). Although an agonist on chemoattractant receptor–homologous molecule expressed on Th2 cells (CRTH2) did not have any effect, the prostanoid DP–receptor agonist BW245C (0.01 – 1 μM) suppressed TGF-β–induced collagen secretion. PGD2 and BW245C significantly increased intracellular cAMP level. One-hour pretreatment with forskolin (0.1 – 10 μM), dibutyryl-cAMP (0.01 – 1 mM), and the protein kinase A (PKA)-activator N6-phenyl-cyclic AMP (100 μM) significantly reduced TGF-β–induced collagen secretion, while exchange protein activated by cAMP (Epac) activator 8-bromo-2′-Omethyladenosine-3′,5′-cyclic AMP (10 μM) did not affect collagen deposition. These results suggest that PGD2 inhibits TGF-β–induced collagen secretion via intracellular cAMP accumulation through activating DP receptor. Keywords:: prostaglandin D2, lung fibroblast, TGF-β, collagen secretion, prostanoid DP receptor

Pulmonary fibrosis is a progressive and fatal lung disease with limited therapeutic options. Although it is well known that lipid mediator prostaglandins are involved in the development of pulmonary fibrosis, the role of prostaglandin D2 (PGD2) remains unknown. Here, we investigated whether genetic disruption of hematopoietic PGD synthase (H-PGDS) affects the bleomycin-induced lung inflammation and pulmonary fibrosis in mouse. Compared with H-PGDS naive (WT) mice, H-PGDS-deficient mice (H-PGDS-/-) represented increased collagen deposition in lungs 14 days after the bleomycin injection. The enhanced fibrotic response was accompanied by an increased mRNA expression of inflammatory mediators, including tumor necrosis factor-α, monocyte chemoattractant protein-1, and cyclooxygenase-2 on day 3. H-PGDS deficiency also increased vascular permeability on day 3 and infiltration of neutrophils and macrophages in lungs on day 3 and 7. Immunostaining showed that the neutrophils and macrophages expressed H-PGDS, and its mRNA expression was increased on day 3and 7 in WT lungs. These observations suggest that H-PGDS-derived PGD2 plays a protective role in bleomycin-induced lung inflammation and pulmonary fibrosis.

Farnesoid X receptor (FXR), a member of the nuclear receptor superfamily that is highly expressed in enterohepatic tissue, is implicated in bile acid, lipid, and glucose metabolisms. Although recent studies showed that FXR is also expressed in vascular endothelial cells and smooth muscle cells, its physiological and/or pathological roles in vasculature tissue remain unknown. The aim of this study is to examine the chronic effect of synthetic FXR agonist GW4064 on vascular contraction and endothelium-dependent relaxation using tissue culture procedure. In cultured rabbit mesenteric arteries, the treatment with 0.1–10 μM GW4064 for 7 days did not influence vascular contractility induced by high K+(15–65 mM), norepinephrine (0.1–100 μM), and endothelin-1 (0.1–100 nM). However, the chronic treatment with GW4064 (1–10 μM for 7 days) dose dependently impaired endothelium-dependent relaxation induced by substance P (0.1–30 nM). In hematoxylin-eosin cross sectioning and en face immunostaining, GW4064 had no effects on the morphology of endothelial and smooth muscle cells. In endothelium-denuded arteries treated with GW4064 (1–10 μM) for 7 days, 3 nM–100 μM sodium nitroprusside-induced vasorelaxation, but not membrane-permeable cGMP analog 8-bromoguanosine-cGMP (8-Br-cGMP; 1–100 μM)-induced vasorelaxation, was significantly impaired. In these GW4064-treated arteries, 1 μM sodium nitroprusside-induced intracellular cGMP elevations were impaired. In RT-PCR, any changes were detected in mRNA expression level of α1- and β1-subunit of soluble guanylyl cyclase. These results suggest that chronic stimulation of FXR impairs endothelium-dependent relaxation, which is due to decreased sensitivity of smooth muscle cells to nitric oxide.

Cancer stem cells (CSCs) are considered to play an important role in cancer therapy resistance, metastasis, and recurrence. Wnt/β-catenin is one of key signaling pathways that confer cancer stemness properties to a portion of the heterogeneous cancer cell population. Dysregulation of the Wnt/β-catenin pathway may lead to accumulation of β-catenin and aberrant activation of its target gene transcription. However, it has been proven difficult to design a therapeutic targeting β-catenin. Identification of a drugguble target for the β-catenin pathway is highly desirable given the importance of β-catenin in promoting cancer stemness. Here, we demonstrate that Serine Threonine Kinase 17A (STK17A), a novel member of the death-associated protein family of serine/threonine kinases, promotes cancer stemness phenotypes via β-catenin. Over-expression of STK17A resulted in increased anchorage-independent spherogenesis and invasive phenotype of cancer cells, whereas STK17A gene silencing reduced these malignant phenotypes. Overexpression of STK17A led to increased transcriptional activity of β-catenin and upregulation of its target genes. These changes were accompanied by increased phosphorylation of β-catenin at ser675. Cell-free in vitro kinase assay revealed that STK17A directly phosphorylates β-catenin at ser675 in an ATP-dependent manner. Moreover, BBI-503 (Amcasertib), a first-in-class cancer stemness kinase inhibitor, was found to potently inhibit STK17A, leading to inhibition of β-catenin phosphorylation at ser675. These results suggest that inhibition of STK17A is a promising approach for targeting cancer stemness. Citation Format: Taiki Kida, Harry A. Rogoff, Chiang J. Li. STK17A, a novel serine threonine kinase, promotes cancer stemness phenotypes by phosphorylating β-catenin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-023. doi:10.1158/1538-7445.AM2017-LB-023

Objective— Although stromal cell–derived factor (SDF)-1αis well known to modulate the mobilization of hematopoietic stem cells and endothelial progenitor cells, its effects on some pre-existing vascular functions remain unknown. We have investigated here the role of SDF-1αsignaling in endothelial barrier function. Approach and Results— Treatment with SDF-1α elevated transendothelial electrical resistance and inhibited the dextran hyperpermeability elicited by thrombin in bovine aortic endothelial cells, both indicating an increase in endothelial barrier function. SDF-1α binds to 2 receptors, C-X-C chemokine receptor types 4 and 7 (CXCR4 and CXCR7). Pretreatment with a CXCR4 antagonist or CXCR4 gene depletion by small interfering RNA (siRNA) eliminated SDF-1α–induced endothelial barrier enhancement. In contrast, CXCR7 antagonist or CXCR7 gene depletion by siRNA did not influence SDF-1α–induced barrier enhancement. Pretreatment with a Gi-protein inhibitor, a phosphoinositide 3-kinase (PI3K) inhibitor, or PI3K p110γsubunit gene depletion by siRNA also inhibited SDF-1α–induced barrier enhancement significantly. Western blot analysis revealed that SDF-1α phosphorylated Akt Ser473 in endothelial cells, suggesting PI3K activation. Immunostaining showed that treatment with SDF-1αformed a cortical actin rim, which was accompanied by Rac1 activation. In vivo, SDF-1αinhibited croton oil–induced vascular leakage indexed by dye extravasation, which is attenuated by a pretreatment with a CXCR4 antagonist. Conclusions— We have identified SDF-1α as a novel suppressor of endothelial permeability. Specifically, SDF-1α stimulates the CXCR4/PI3K/Rac1 signaling pathway and the subsequent cytoskeletal rearrangement.

Bile acids are end products of cholesterol metabolism, and they constantly exist at high concentrations in the blood. Since vascular endothelial cells express G protein–coupled bile acid receptor (GPBAR), bile acids potentially modulate endothelial function. Here, we investigated whether and how GPBAR agonism affects endothelial barrier function. In bovine aortic endothelial cells (BAECs), treatment with a GPBAR agonist, taurolithocholic acid (TLCA) increased the transendothelial electrical resistance. In addition, TLCA suppressed the thrombin-induced dextran infiltration through the endothelial monolayer. Knockdown of GPBAR abolished the inhibitory effect of TLCA on hyperpermeability. These results indicate that stimulation of GPBAR enhances endothelial barrier function. TLCA increased intracellular cAMP production in BAECs. Inhibition of protein kinase A (PKA) or Rac1 significantly attenuated the TLCA-induced endothelial barrier protection. TLCA induced cortical actin polymerization, which was attenuated by a Rac1 inhibitor. In vivo, local administration of TLCA into the mouse ear significantly inhibited vascular leakage and edema formation induced by croton oil or vascular endothelial growth factor. These results indicate that stimulation of GPBAR enhances endothelial barrier function by cAMP/PKA/Rac1-dependent cytoskeletal rearrangement.

Objective— TGR5 is a G-protein–coupled receptor for bile acids. So far, little is known about the function of TGR5 in vascular endothelial cells. Approach and Results— In bovine aortic endothelial cells, treatment with a bile acid having a high affinity to TGR5, taurolithocholic acid (TLCA), significantly increased NO production. This effect was abolished by small interfering RNA–mediated depletion of TGR5. TLCA-induced NO production was also observed in human umbilical vein endothelial cells measured via intracellular cGMP accumulation. TLCA increased endothelial NO synthase ser1177 phosphorylation in human umbilical vein endothelial cells. This response was accompanied by increased Akt ser473 phosphorylation and intracellular Ca 2+ . Inhibition of these signals significantly decreased TLCA-induced NO production. We next examined whether TGR5-mediated NO production affects inflammatory responses of endothelial cells. In human umbilical vein endothelial cells, TLCA significantly reduced tumor necrosis factor-α–induced adhesion of monocytes, vascular cell adhesion molecule-1 expression, and activation of nuclear factor-κB. TLCA also inhibited lipopolysaccharide-induced monocyte adhesion to mesenteric venules in vivo. These inhibitory effects of TLCA were abrogated by NO synthase inhibition. Conclusions— TGR5 agonism induces NO production via Akt activation and intracellular Ca 2+ increase in vascular endothelial cells, and this function inhibits monocyte adhesion in response to inflammatory stimuli.

Prostaglandin E₂ (PGE₂) is a major prostanoid produced under inflammatory situations. There have been controversial reports showing contractile or relaxant effect of PGE₂ on vascular tone in various types of blood vessels. Thus, it is still elusive whether and how PGE₂ modulates vascular tone. We here assessed the effects of PGE₂ on vascular contractility using different types of vasculatures isolated form rat. In endothelium-denuded aortas and mesenteric arteries, PGE₂ (1 nM-10 μM) concentration-dependently enhanced the contraction elicited by K(+) (35.4 mM) or norepinephrine (10 nM). In pulmonary arteries, PGE₂ did not alter the both-induced contraction. Tail arteries were relaxed by a low dose of PGE₂ (1-100 nM), but this response shifted to contraction by the higher dose of PGE₂ (300 nM-10 μM). There are four types of PGE₂ receptors EP1-4. RT-PCR showed that aortas and mesenteric arteries abundantly expressed EP3, while tail arteries abundantly expressed EP4. We next revealed that selective EP3 agonism enhanced the contraction in mesenteric arteries, whereas EP4 agonism induced relaxation in tail arteries. Taken together, PGE₂ causes different contractile responses depending on the type of vascular bed. This phenomenon may be due to the difference in expression pattern and activity of EP receptors.

Objective TGR5, a membrane-bound, G-protein-coupled receptor for bile acids, is known to be involved in regulation of energy homeostasis and inflammation. However, little is known about the function of TGR5 in vascular endothelial cells. In the present study, we examined whether TGR5 agonism represents anti-inflammatory effects in vascular endothelial cells focusing on nitric oxide (NO) production. Methods and Results In human umbilical vein endothelial cells (HUVECs), treatment with taurolithocholic acid (TLCA), which has the highest affinity to TGR5 among various bile acids, significantly reduced tumor necrosis factor (TNF)-α-induced vascular cell adhesion molecule (VCAM)-1 protein expression and adhesion of human monocytes, U937. These effects were abrogated by a NO synthase (NOS) inhibitor, N G -Monomethyl-L-arginine (L-NMMA). In bovine aortic endothelial cells (BAECs), treatment with TLCA as well as lithocholic acid, which also has high affinity to TGR5, significantly increased the NO production. In contrast, deoxycholic acid and chenodeoxycholic acid, which possess low affinity to TGR5, did not affect the NO production. Gene depletion of TGR5 by siRNA transfection abolished TLCA-induced NO production in BAECs. TLCA-induced NO production was also observed in HUVECs measured as intracellular cGMP accumulation. We next investigated the signal pathways responsible for the TLCA-induced NO production in endothelial cells. Treatment with TLCA increased endothelial NOS (eNOS) ser1177 phosphorylation in HUVECs. This response was accompanied by increased Akt ser473 phosphorylation and intracellular Ca 2+ ([Ca 2+ ] i ). Treatment with phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, or blockade of calcium channel with La 3+ , significantly decreased TLCA-induced eNOS ser1177 phosphorylation and subsequent NO production. Conclusion These results indicate that TGR5 agonism can mediate anti-inflammatory responses by suppressing VCAM-1 expression and monocytes adhesion to endothelial cells. This function is dependent on NO production via Akt activation and [Ca 2+ ] i increase.

Objective In this study, we examined the synergistic effects of the two potent pathogenic factors, platelet-derived growth factor-BB (PDGF-BB) and endothelin-1 (ET-1) to induce vascular hyperplasia using ex vivo organ-culture system. Methods and results In organ-cultured rat tail arteries, concomitant treatment with 100ng/ml PDGF-BB and 300nM ET-1 for 4 days induced medial hyperplasia with increased smooth muscle cell proliferation. Concomitant treatment with PDGF-BB (10–300nM) and ET-1 (30nM–1μM) dose-dependently suppressed contractile responses to high K + and norepinephrine. This dyscontractility was accompanied by decreased α-actin protein expression. In all series of experiments, concomitant treatment with PDGF-BB and ET-1 exhibited stronger effects than sole treatment with PDGF-BB (100ng/ml) or ET-1 (300nM). Western blot analysis revealed that concomitant treatment with PDGF-BB and ET-1 synergistically phosphorylated extracellular signal-regulated kinase 1 and 2 (ERK1/2), Akt, and a downstream target of mammalian target of rapamycin (mTOR), p70 ribosomal S6 kinase in cultured artery. Consistently, a MAPK/ERK kinase (MEK) inhibitor, PD98059 (30μM), a phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, and an mTOR inhibitor, rapamycin (30nM), partially restored PDGF-BB and ET-1-induced hyperplastic changes. Conclusions We evidenced for the first time at tissue level that PDGF-BB and ET-1 synergistically accelerate vascular smooth muscle hyperplastic changes and lose its contractility, at least partially through ERK1/2, Akt, and mTOR activation.