Mehari Endale

Quercetin disrupts tyrosine-phosphorylated phosphatidylinositol 3-kinase and myeloid differentiation factor-88 association, and inhibits MAPK/AP-1 and IKK/NF-κB-induced inflammatory mediators production in RAW 264.7 cells

Quercetin is a major bioflavonoid widely present in fruits and vegetables. It exhibits anti-inflammatory, anti-tumor, antioxidant properties and reduces cardiovascular disease risks. However, the molecular mechanism of action against inflammation in RAW 264.7 cells is only partially explored. Quercetin effect on LPS-induced gene and protein expressions of inflammatory mediators and cytokines were determined. Moreover, involvement of heme-oxygenase-1, protein kinases, adaptor proteins and transcription factors in molecular mechanism of quercetin action against inflammation were examined. Quercetin inhibited LPS-induced NO, PGE₂, iNOS, COX-2, TNF-α, IL-1β, IL-6 and GM-CSF mRNA and protein expressions while it promoted HO-1 induction in a dose- and time-dependent manner. It also suppressed I-κB-phosphorylation, NF-κB translocation, AP-1 and NF-κB-DNA-binding and reporter gene transcription. Quercetin attenuated p38(MAPK) and JNK1/2 but not ERK1/2 activations and this effect was further confirmed by SB203580 and SP600125-mediated suppressions of HO-1, iNOS, and COX-2 protein expressions. Moreover, quercetin arrested Src, PI3K, PDK1 and Akt activation in a time- and dose-dependent manner, which was comparable to PP2 and LY294002 inhibition of Src, PI3K/Akt and iNOS expressions. Quercetin further arrested Src and Syk tyrosine phosphorylations and their kinase activities followed by inhibition of PI3K tyrosine phosphorylation. Moreover, quercetin disrupted LPS-induced p85 association to TLR4/MyD88 complex and it then limited activation of IRAK1, TRAF6 and TAK1 with a subsequent reduction in p38 and JNK activations, and suppression in IKKα/β-mediated I-κB phosphorylation. Quercetin limits LPS-induced inflammation via inhibition of Src- and Syk-mediated PI3K-(p85) tyrosine phosphorylation and subsequent TLR4/MyD88/PI3K complex formation that limits activation of downstream signaling pathways.

Ginsenoside-Rp1 inhibits platelet activation and thrombus formation via impaired glycoprotein VI signalling pathway, tyrosine phosphorylation and MAPK activation

Ginsenosides are the main constituents for the pharmacological effects of Panax ginseng. Such effects of ginsenosides including cardioprotective and anti‐platelet activities have shown stability and bioavailability limitations. However, information on the anti‐platelet activity of ginsenoside‐Rp1 (G‐Rp1), a stable derivative of ginsenoside‐Rg3, is scarce. We examined the ability of G‐Rp1 to modulate agonist‐induced platelet activation.

A noble function of BAY 11-7082: Inhibition of platelet aggregation mediated by an elevated cAMP-induced VASP, and decreased ERK2/JNK1 phosphorylations.

Platelets, though anucleated, possess several transcription factors, including NF-kappaB, that exert non-genomic functions regulating platelet activation. Since platelets have not only been recognized as central players of homeostasis, but also participated in pathological conditions such as thrombosis, atherosclerosis, and inflammation, we examined rat platelet NF-kappaB expression and evaluated the effects of anti-inflammatory drug BAY 11-7082, an inhibitor of NF-kappaB activation, in platelet physiology. Western blotting revealed that rat platelets express NF-kappaB. BAY 11-7082, dose dependently, inhibited collagen- or thrombin-induced-platelet aggregation. ATP release, TXB(2) formation, P-selectin expression, and intercellular Ca(2+) concentration activated by collagen were reduced in BAY 11-7082-treated platelets. BAY 11-7082 elevated intracellular levels of cAMP, but not cGMP, and its co-incubation with cAMP-activating agent (forskolin) or its hydrolyzing enzyme inhibitor (3-isobutyl-1-methylxanthine, IBMX), synergistically inhibited collagen-induced-platelet aggregation. In addition, vasodilator-stimulated-phosphoprotein (VASP) phosphorylation was enhanced in BAY 11-7082-treated platelets, which was partially inhibited by a protein kinase A (PKA) inhibitor, H-89. Moreover, while p38 mitogen-activated protein kinase (MAPK) was not affected, BAY 11-7082 attenuated c-Jun N-terminal kinase 1 (JNK1) and extracellular-signal-regulated protein kinase 2 (ERK2) phosphorylations. In conclusion, BAY 11-7082 inhibits platelet activation, granule secretion, and aggregation, and that this effect is mediated by inhibition of JNK1 and ERK2 phosphorylations, and partially by stimulation of cAMP-dependent PKA VASP phosphorylation. The ability of BAY 11-7082 to inhibit platelet function might be relevant in cases involving aberrant platelet activation where the drug is considered as anti-atherothrombosis, and anti-inflammatory therapy.

Diversity of Interstitial Lung Fibroblasts Is Regulated by Platelet-Derived Growth Factor Receptor α Kinase Activity

Epithelial-mesenchymal cell interactions and factors that control normal lung development are key players in lung injury, repair, and fibrosis. A number of studies have investigated the roles and sources of epithelial progenitors during lung regeneration; such information, however, is limited in lung fibroblasts. Thus, understanding the origin, phenotype, and roles of fibroblast progenitors in lung development, repair, and regeneration helps address these limitations. Using a combination of platelet-derived growth factor receptor α-green fluorescent protein (PDGFRα-GFP) reporter mice, microarray, real-time polymerase chain reaction, flow cytometry, and immunofluorescence, we characterized two distinct interstitial resident fibroblasts, myo- and matrix fibroblasts, and identified a role for PDGFRα kinase activity in regulating their activation during lung regeneration. Transcriptional profiling of the two populations revealed a myo- and matrix fibroblast gene signature. Differences in proliferation, smooth muscle actin induction, and lipid content in the two subpopulations of PDGFRα-expressing fibroblasts during alveolar regeneration were observed. Although CD140α(+)CD29(+) cells behaved as myofibroblasts, CD140α(+)CD34(+) appeared as matrix and/or lipofibroblasts. Gain or loss of PDGFRα kinase activity using the inhibitor nilotinib and a dominant-active PDGFRα-D842V mutation revealed that PDGFRα was important for matrix fibroblast differentiation. We demonstrated that PDGFRα signaling promotes alveolar septation by regulating fibroblast activation and matrix fibroblast differentiation, whereas myofibroblast differentiation was largely PDGFRα independent. These studies provide evidence for the phenotypic and functional diversity as well as the extent of specificity of interstitial resident fibroblasts differentiation during regeneration after partial pneumonectomy.

Dual Roles of Quercetin in Platelets: Phosphoinositide-3-Kinase and MAP Kinases Inhibition, and cAMP-Dependent Vasodilator-Stimulated Phosphoprotein Stimulation

Background. Progressive diseases including cancer, metabolic, and cardiovascular disorders are marked by platelet activation and chronic inflammation. Studies suggest that dietary flavonoids such as quercetin possess antioxidant, anti-inflammatory, and antiplatelet properties, which could prevent various chronic diseases including atherosclerosis and thrombosis. However, the mechanism and the signaling pathway that links quercetins antiplatelet activity with its anti-inflammatory property is limited and thus further exploration is required. The aim of this paper was to examine the link between antiplatelet and anti-inflammatory roles of quercetin in agonist-induced platelet activation. Methods. Quercetin effects on agonist-activated platelet-aggregation, granule-secretion, [Ca2+]i, and glycoprotein-IIb/IIIa activation were examined. Its effects on PI3K/Akt, VASP, and MAPK phosphorylations were also studied on collaged-activated platelets. Results. Quercetin dose dependently suppressed collagen, thrombin, or ADP-induced platelet aggregation. It significantly inhibited collagen-induced ATP release, P-selectin expression, [Ca2+]i mobilization, integrin-α IIb β 3 activation, and augmented cAMP and VASP levels. Moreover, quercetin attenuated PI3K, Akt, ERK2, JNK1, and p38 MAPK activations, which were supported by platelet-aggregation inhibition with the respective kinase inhibitors. Conclusion. Quercetin-mediated antiplatelet activity involves PI3K/Akt inactivation, cAMP elevation, and VASP stimulation that, in turn, suppresses MAPK phosphorylations. This result suggests quercetin may have a potential to treat cardiovascular diseases involving aberrant platelet activation and inflammation.

Inhibitory effects of Bulnesia sarmienti aqueous extract on agonist-induced platelet activation and thrombus formation involves mitogen-activated protein kinases

ETHNOPHARMACOLOGICAL RELEVANCEnB. sarmienti has long been recognized in folk medicine as a medicinal plant with various medicinal uses. Traditionally, it has been appreciated for the skin-healing properties of its essence. The bark has also been employed to treat stomach and cardiovascular disorders and reported to have antitumor, antioxidant and anti-inflammatory activities. However, information on its antiplatelet activity is limited.nnnAIM OF THE STUDYnTo examined the effects of B. sarmienti aqueous extract (BSAE) in platelet physiology.nnnMATERIALS AND METHODSnThe anti-platelet activity of BSAE was studied using rat platelets for in vitro determination of the extract effect on agonist-induced platelet aggregation, ATP secretion, [Ca(2+)](i) mobilization and MAP kinase phosphorylation. The extract in vivo effects was also examined in arterio-venous shunt thrombus formation in rats, and tail bleeding time in mice.nnnRESULTnHPLC chromatographic analysis revealed that B. sarmienti extract contained (+)-catechin (C), (-)-epigallocatechin (EGC), (-)-epicatechin (EC), and (-)-epicatechin gallate (ECG). BSAE, significantly and dose dependently, inhibited collagen, thrombin, or ADP-induced platelet aggregation. The 50 percent inhibitory concentrations (IC(50)) of the extract for collagen, thrombin and ADP-induced platelet aggregation were 45.3+/-2.6, 100+/-5.6 and 110+/-4.6 microg/ml, respectively. Collagen activated ATP release and thrombin-induced intracellular Ca(2+) concentration were reduced in BSAE-treated platelets. In addition, the extract in vivo activity showed that BSAE at 100 mg/kg significantly attenuated thrombus formation in rat extracorporeal shunt model while mice tail bleeding time was not affected. Moreover, BSAE attenuated p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N-terminal kinase 1 (JNK1) and extracellular-signal-regulated protein kinase 2 (ERK2) phosphorylations.nnnCONCLUSIONnBSAE inhibits platelet activation, granule secretion, aggregation, and thrombus formation without affecting bleeding time, and that this effect is mediated by inhibition of P38, JNK1 and ERK2 phosphorylations. The ability of BSAE to inhibit platelet function might be relevant in cases involving aberrant platelet activation where the plant extract could be considered as a candidate to anti-platelet and antithrombotic agent.

Antiplatelet Activity of Phellinus baummii Methanol Extract is Mediated by Cyclic AMP Elevation and Inhibition of Collagen-activated Integrin-αIIbβ3 and MAP Kinase

Phellinus baumii is a mushroom that has been used as folk medicine against various diseases and is reported to have antidiabetic, anticancer, antioxidant, antiinflammatory and antihypertensive activities. However, information on the effects of P. baumii extract in platelet function is limited. Therefore, the aim of this study was to examine the impact of a P. baumii methanol extract (PBME) on platelet activation and to investigate the mechanism behind its antiplatelet activity. PBME effects on agonist‐induced platelet aggregation, granule secretion, [Ca2+]i mobilization, αIIbβ3 activation, cyclic AMP release and mitogen‐activated protein kinase (MAPK) phosphorylations were studied using rat platelets. PBME dose‐dependently inhibited collagen, thrombin and ADP‐induced platelet aggregation with an IC50 of 51.0u2009±u20092.4, 54.0u2009±u20092.1 and 53.0u2009±u20094.3u2009μg/mL, respectively. Likewise, thrombin‐induced [Ca2+]i and collagen‐activated ATP secretions were suppressed in PBME treated platelets. Aggregation and ATP secretion were also markedly attenuated by PBME alone or in combination with PP2 (Src inhibitor) and U‐73122 (PLC inhibitor) in collagen‐stimulated platelets. Besides, PBME treatment elevated basal cyclic AMP levels and inhibited collagen‐induced integrin‐αIIbβ3 activation. Moreover, PBME attenuated extracellular‐signal‐regulated protein kinase 2 (ERK2) and c‐Jun N‐terminal kinase 1 (JNK1) phosphorylations. Further PD98059 (ERK inhibitor) and SP60025 (JNK inhibitor) reduced collagen‐induced platelet aggregation and ATP secretion. In conclusion, the observed PBME antiplatelet activity may be mediated by activation of cyclic AMP and inhibition of ERK2 and JNK1 phosphorylations. Finally, these data suggest that PBME may have therapeutic potential for the treatment of cardiovascular diseases that involve aberrant platelet function. Copyright

Ischemia induces regulator of G protein signaling 2 (RGS2) protein upregulation and enhances apoptosis in astrocytes

Regulator of G protein signaling (RGS) family members, such as RGS2, interact with Galpha subunits of heterotrimeric G proteins, accelerating the rate of GTP hydrolysis and attenuating the intracellular signaling triggered by the G protein-coupled receptor-ligand interaction. They are also reported to regulate G protein-effector interactions and form multiprotein signaling complexes. Ischemic stress-induced changes in RGS2 expression have been described in astrocytes, and these changes are associated with intracellular signaling cascades, suggesting that RGS2 upregulation may be an important mechanism by which astrocytes may regulate RGS2 function in response to physiological stress. However, information on the functional roles of stress-induced modulation of RGS2 protein expression in astrocyte function is limited. We report the role of ischemic stress in RGS2 protein expression in rat C6 astrocytoma cells and primary mouse astrocytes. A marked increase in RGS2 occurred after ischemic stress induced by chemicals (sodium azide and 2-deoxyglucose) or oxygen-glucose deprivation (OGD, real ischemia). RGS2 mRNA expression was markedly enhanced by 1 h of exposure to chemical ischemia or 6 h of OGD followed by 2 or 6 h of recovery, respectively. This enhanced expression in primary astrocytes and C6 cells was restored to baseline levels after 12 h of recovery from chemically induced ischemic stress or 4-6 h of recovery from OGD. RGS2 protein was also significantly expressed at 12-24 h of recovery from ischemic insult. Ischemia-induced RGS2 upregulation was associated with enhanced apoptosis. It significantly increased annexin V-positive cells, cleaved caspase-3, and enhanced DNA ladder formation and cell cycle arrest. However, a small interfering RNA (siRNA)-mediated RGS2 knockdown reversed the apoptotic cell death associated with ischemia-induced RGS2 upregulation. Upregulated RGS2 was significantly inhibited by SB-203580, a p38 MAPK inhibitor. Rottlerin, a potent inhibitor of PKCdelta, completely abrogated the increased RGS2 expression. We also examine whether ischemia-induced RGS2-mediated apoptosis is affected by siRNA-targeted endogenous PKCdelta downregulation or its phosphorylation. Although RGS2 upregulation was not affected, siRNA transfection significantly suppressed endogenous PKCdelta mRNA and protein expressions. Ischemia-induced PKCdelta phosphorylation and caspase-3 cleavage were dose dependently inhibited by PKCdelta knockdown, and this endogenous PKCdelta suppression reversed ischemia-induced annexin V-positive cells. This study suggests that ischemic stress increases RGS2 expression and that this condition contributes to enhanced apoptosis in C6 cells and primary astrocytes. The signaling it follows may involve PKCdelta and p38 MAPK pathways.

Temporal, spatial, and phenotypical changes of PDGFRα expressing fibroblasts during late lung development

Many studies have investigated the source and role of epithelial progenitors during lung development; such information is limited for fibroblast populations and their complex role in the developing lung. In this study, we characterized the spatial location, mRNA expression and Immunophenotyping of PDGFRα+ fibroblasts during sacculation and alveolarization. Confocal microscopy identified spatial association of PDGFRα expressing fibroblasts with proximal epithelial cells of the branching bronchioles and the dilating acinar tubules at E16.5; with distal terminal saccules at E18.5; and with alveolar epithelial cells at PN7 and PN28. Immunohistochemistry for alpha smooth muscle actin revealed that PDGFRα+ fibroblasts contribute to proximal peribronchiolar smooth muscle at E16.5 and to transient distal alveolar myofibroblasts at PN7. Time series RNA-Seq analyses of PDGFRα+ fibroblasts identified differentially expressed genes that, based on gene expression similarity were clustered into 7 major gene expression profile patterns. The presence of myofibroblast and smooth muscle precursors at E16.5 and PN7 was reflected by a two-peak gene expression profile on these days and gene ontology enrichment in muscle contraction. Additional molecular and functional differences between peribronchiolar smooth muscle cells at E16.5 and transient intraseptal myofibroblasts at PN7 were suggested by a single peak in gene expression at PN7 with functional enrichment in cell projection and muscle cell differentiation. Immunophenotyping of subsets of PDGFRα+ fibroblasts by flow cytometry confirmed the predicted increase in proliferation at E16.5 and PN7, and identified subsets of CD29+ myofibroblasts and CD34+ lipofibroblasts. These data can be further mined to develop novel hypotheses and valuable understanding of the molecular and cellular basis of alveolarization.

RGS2-Mediated Intracellular Ca2+ Level Plays a Key Role in the Intracellular Replication of Brucella abortus Within Phagocytes

BACKGROUNDnBrucella abortus can proliferate within professional and nonprofessional phagocytic host cells and thereby successfully bypass the bacteriocidal effects of phagocytes. However, the intracellular survival mechanism and factors of virulence are not fully understood.nnnMETHODSnWe have investigated the role of the regulator of G protein signaling 2 (RGS2), an intracellular calcium ([Ca(2+)](i)) regulator of the host cell, in the intracellular survival of B. abortus within phagocytes.nnnRESULTSnB. abortus infection markedly induced RGS2 messenger RNA expression in early phase and increased the [Ca(2+)](i) level up to 24 hours postinfection within macrophages from wild-type mice. The [Ca(2+)](i) level, however, was not influenced by B. abortus infection within macrophages from RGS2-deficient mice. Furthermore, B. abortus survival was reduced within RGS2-deficient macrophages, and hence bacterial proliferation was inhibited in RGS2-deficient mice. Moreover, treatment with the Ca(2+) chelator ethylenediaminetetraacetic acid (EDTA) or 1,2-bis-(2-amino-phenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and the L-type Ca(2+) channel-blocking agent nifedipine or genistein also showed a reduced intracellular replication of B. abortus within macrophages.nnnCONCLUSIONnThese results indicate that B. abortus infection induces host RGS2 expression and that up-regulation of [Ca(2+)](i) levels is an essential factor for the intracellular survival of B. abortus within phagocytes.