Kink-band formation in the directionally-solidified Mg/LPSO two-phase alloys.

The variation in the mechanical properties with the volume fraction of the long-period stacking ordered (LPSO) phase in directionally solidified (DS) Mg/LPSO two-phase alloys was examined. Unexpectedly, the yield stress of the DS alloys increases non-monotonically with an increase in the volume fraction of the LPSO phase. The LPSO phase is considered an effective strengthening phase in Mg alloys, when the stress is applied parallel to the growth direction. Nevertheless, the highest strength was obtained in alloys with 61-86 vol.% of the LPSO phase, which was considerably higher than that in the LPSO single-phase alloy. It was clarified that this complicated variation in the yield stress was generated from the change in the formation stress of kink bands, which varied with the thickness of the LPSO-phase grains. Furthermore, the coexistence of Mg in the LPSO phase alloy induced the homogeneous formation of kink bands in the alloys, leading to the enhancement of the 'kink-band strengthening'. The results demonstrated that microstructural control is significantly important in Mg/LPSO two-phase alloys, in which both phases exhibit strong plastic anisotropy, to realize the maximum mechanical properties.

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)-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.

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.

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 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.

Diverse effects of prostaglandin E2 on vascular contractility.

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.

Prostaglandin D2 inhibits collagen secretion from lung fibroblasts by activating the DP receptor.

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-ß (TGF-ß). Although an agonist on chemoattractant receptorhomologous molecule expressed on Th2 cells (CRTH2) did not have any effect, the prostanoid DP-receptor agonist BW245C (0.01 - 1 µM) suppressed TGF-ß-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 N(6)-phenyl-cyclic AMP (100 µM) significantly reduced TGF-ß-induced collagen secretion, while exchange protein activated by cAMP (Epac) activator 8-bromo-2'-O-methyladenosine-3',5'-cyclic AMP (10 µM) did not affect collagen deposition. These results suggest that PGD2 inhibits TGF-ß-induced collagen secretion via intracellular cAMP accumulation through activating DP receptor.

The profile of urinary lipid metabolites in healthy dogs.

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.

Urinary lipid production profile in canine patients with splenic mass.

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) E2, F2α, and D2, 8-iso-PGF3α, lyso-platelet activating factor, and 14,15-leukotrien C4 significantly increased in urine samples of dogs with splenic mass compared to that of healthy dogs. These observations may reflect general inflammatory responses and will help better understanding of the canine splenic mass.

Comprehensive profiling of lipid metabolites in urine of canine patients with liver mass.

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 E2 and/or PGF2α. We demonstrated that our approach of profiling lipid metabolites in the urine is useful in gaining insights into the disease. These findings may also have an application as a screening test or a diagnosis tool for canine liver mass.

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.

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.

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.