Taiki Kida

Bile Acid Receptor TGR5 Agonism Induces NO Production and Reduces Monocyte Adhesion in Vascular Endothelial Cells

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 synthaseser1177 phosphorylation in human umbilical vein endothelial cells. This response was accompanied by increased Aktser473 phosphorylation and intracellular Ca2+. 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-&agr;–induced adhesion of monocytes, vascular cell adhesion molecule-1 expression, and activation of nuclear factor-&kgr;B. 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 Ca2+ increase in vascular endothelial cells, and this function inhibits monocyte adhesion in response to inflammatory stimuli.

Chronic stimulation of farnesoid X receptor impairs nitric oxide sensitivity of vascular smooth muscle

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 microM GW4064 for 7 days did not influence vascular contractility induced by high K(+) (15-65 mM), norepinephrine (0.1-100 microM), and endothelin-1 (0.1-100 nM). However, the chronic treatment with GW4064 (1-10 microM 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 microM) for 7 days, 3 nM-100 microM sodium nitroprusside-induced vasorelaxation, but not membrane-permeable cGMP analog 8-bromoguanosine-cGMP (8-Br-cGMP; 1-100 microM)-induced vasorelaxation, was significantly impaired. In these GW4064-treated arteries, 1 muM sodium nitroprusside-induced intracellular cGMP elevations were impaired. In RT-PCR, any changes were detected in mRNA expression level of alpha(1)- and beta(1)-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.

Multiple roles of the PGE2-EP receptor signal in vascular permeability

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.

Stromal Cell–Derived Factor-1α/C-X-C Chemokine Receptor Type 4 Axis Promotes Endothelial Cell Barrier Integrity via Phosphoinositide 3-Kinase and Rac1 Activation

Objective— Although stromal cell–derived factor (SDF)-1&agr;is 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-1&agr;signaling in endothelial barrier function. Approach and Results— Treatment with SDF-1&agr; 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-1&agr; 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-1&agr;–induced endothelial barrier enhancement. In contrast, CXCR7 antagonist or CXCR7 gene depletion by siRNA did not influence SDF-1&agr;–induced barrier enhancement. Pretreatment with a Gi-protein inhibitor, a phosphoinositide 3-kinase (PI3K) inhibitor, or PI3K p110&ggr;subunit gene depletion by siRNA also inhibited SDF-1&agr;–induced barrier enhancement significantly. Western blot analysis revealed that SDF-1&agr; phosphorylated AktSer473 in endothelial cells, suggesting PI3K activation. Immunostaining showed that treatment with SDF-1&agr;formed a cortical actin rim, which was accompanied by Rac1 activation. In vivo, SDF-1&agr;inhibited croton oil–induced vascular leakage indexed by dye extravasation, which is attenuated by a pretreatment with a CXCR4 antagonist. Conclusions— We have identified SDF-1&agr; as a novel suppressor of endothelial permeability. Specifically, SDF-1&agr; stimulates the CXCR4/PI3K/Rac1 signaling pathway and the subsequent cytoskeletal rearrangement.

Chronic treatment with PDGF-BB and endothelin-1 synergistically induces vascular hyperplasia and loss of contractility in organ-cultured rat tail artery

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 100 ng/ml PDGF-BB and 300 nM ET-1 for 4 days induced medial hyperplasia with increased smooth muscle cell proliferation. Concomitant treatment with PDGF-BB (10-300 nM) and ET-1 (30 nM-1 μM) 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 (100 ng/ml) or ET-1 (300 nM). 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 (30 μM), a phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, and an mTOR inhibitor, rapamycin (30 nM), 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.

Prostaglandin D2 Attenuates Bleomycin-Induced Lung Inflammation and Pulmonary Fibrosis.

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 naïve (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.

Stimulation of G Protein–Coupled Bile Acid Receptor Enhances Vascular Endothelial Barrier Function via Activation of Protein Kinase A and Rac1

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.