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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.

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.

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.

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.

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.