Nitric oxide (NO) by activating soluble guanylyl cyclase (sGC) is involved in vascular homeostasis via induction of smooth muscle relaxation. In cardiovascular diseases (CVDs), endothelial ...dysfunction with altered vascular reactivity is mostly attributed to decreased NO bioavailability via oxidative stress. However, in several studies, relaxation to NO is only partially restored by exogenous NO donors, suggesting sGC impairment. Conflicting results have been reported regarding the nature of this impairment, ranging from decreased expression of one or both subunits of sGC to heme oxidation. We showed that sGC activity is impaired by thiol S-nitrosation. Recently, angiotensin II (ANG II) chronic treatment, which induces hypertension, was shown to generate nitrosative stress in addition to oxidative stress. We hypothesized that S-nitrosation of sGC occurs in ANG II-induced hypertension, thereby leading to desensitization of sGC to NO hence vascular dysfunction. As expected, ANG II infusion increases blood pressure, aorta remodeling, and protein S-nitrosation. Intravital microscopy indicated that cremaster arterioles are resistant to NO-induced vasodilation in vivo in anesthetized ANG II-treated rats. Concomitantly, NO-induced cGMP production decreases, which correlated with S-nitrosation of sGC in hypertensive rats. This study suggests that S-nitrosation of sGC by ANG II contributes to vascular dysfunction. This was confirmed in vitro by using A7r5 smooth muscle cells infected with adenoviruses expressing sGC or cysteine mutants: ANG II decreases NO-stimulated activity in the wild-type but not in one mutant, C516A. This result indicates that cysteine 516 of sGC mediates ANG II-induced desensitization to NO in cells.
Soluble guanylyl cyclase (sGC), a cyclic guanosine 5'-monophosphate-generating enzyme, regulates smooth muscle tone and exerts antiinflammatory effects in animal models of asthma and acute lung ...injury. In chronic obstructive pulmonary disease (COPD), primarily caused by cigarette smoke (CS), lung inflammation persists and smooth muscle tone remains elevated, despite ample amounts of nitric oxide that could activate sGC.
To determine the expression and function of sGC in patients with COPD and in a murine model of COPD.
Expression of sGCα1, α2, and β1 subunits was examined in lungs of never-smokers, smokers without airflow limitation, and patients with COPD; and in C57BL/6 mice after 3 days, 4 weeks, and 24 weeks of CS exposure. The functional role of sGC was investigated in vivo by measuring bronchial responsiveness to serotonin in mice using genetic and pharmacologic approaches.
Pulmonary expression of sGC, both at mRNA and protein level, was decreased in smokers without airflow limitation and in patients with COPD, and correlated with disease severity (FEV1%). In mice, exposure to CS reduced sGC, cyclic guanosine 5'-monophosphate levels, and protein kinase G activity. sGCα1(-/-) mice exposed to CS exhibited bronchial hyperresponsiveness to serotonin. Activation of sGC by BAY 58-2667 restored the sGC signaling and attenuated bronchial hyperresponsiveness in CS-exposed mice.
Down-regulation of sGC because of CS exposure might contribute to airflow limitation in COPD.
A productive angiogenic response must couple to the survival machinery of endothelial cells to preserve the integrity of newly formed vessels. Angiopoietin-1 (Ang-1) is an endothelium-specific ligand ...essential for embryonic vascular stabilization, branching morphogenesis, and post-natal angiogenesis, but its contribution to endothelial cell survival has not been completely elucidated. Here we show that Ang-1 acting via the Tie 2 receptor induces phosphorylation of the survival serine-threonine kinase, Akt (or protein kinase B). This is associated with up-regulation of the apoptosis inhibitor, survivin, in endothelial cells and protection of endothelium from death-inducing stimuli. Moreover, dominant negative survivin negates the ability of Ang-1 to protect cells from undergoing apoptosis. The activation of anti-apoptotic pathways mediated by Akt and survivin in endothelial cells may contribute to Ang-1 stabilization of vascular structures during angiogenesis, in vivo.
Flavonoids are naturally occurring polyphenolic compounds with a wide distribution throughout the plant kingdom. In the present study, we compared the ability of several flavonoids to modulate the ...production of proinflammatory molecules from lipopolysaccharide (LPS)-stimulated macrophages and investigated their mechanism(s) of action. Pretreatment of RAW 264.7 with luteolin, luteolin-7-glucoside, quercetin, and the isoflavonoid genistein inhibited both the LPS-stimulated TNF-alpha and interleukin-6 release, whereas eriodictyol and hesperetin only inhibited TNF-alpha release. From the compounds tested luteolin and quercetin were the most potent in inhibiting cytokine production with an IC(50) of less than 1 and 5 microM for TNF-alpha release, respectively. To determine the mechanisms by which flavonoids inhibit LPS signaling, we used luteolin and determined its ability to interfere with total protein tyrosine phosphorylation as well as Akt phosphorylation and nuclear factor-kappaB activation. Pretreatment of the cells with luteolin attenuated LPS-induced tyrosine phosphorylation of many discrete proteins. Moreover, luteolin inhibited LPS-induced phosphorylation of Akt. Treatment of macrophages with LPS resulted in increased IkappaB-alpha phosphorylation and reduced the levels of IkappaB-alpha. Pretreatment of cells with luteolin abolished the effects of LPS on IkappaB-alpha. To determine the functional relevance of the phosphorylation events observed with IkappaB-alpha, macrophages were transfected either with a control vector or a vector coding for the luciferase reporter gene under the control of kappaB cis-acting elements. Incubation of transfected RAW 264.7 cells with LPS increased luciferase activity in a luteolin-sensitive manner. We conclude that luteolin inhibits protein tyrosine phosphorylation, nuclear factor-kappaB-mediated gene expression and proinflammatory cytokine production in murine macrophages.
Heat-shock protein 90 (Hsp90) coordinates the trafficking and regulation of diverse signalling proteins, but its precise role in regulating specific cellular targets is not known,. Here we show that ...Hsp90 associates with endothelial nitric oxide synthase (eNOS) and is rapidly recruited to the eNOS complex by agonists that stimulate production of nitric oxide, namely vascular endothelial growth factor, histamine and fluid shear stress. Moreover, the binding of Hsp90 to eNOS enhances the activation of eNOS. Inhibition of signalling through Hsp90 attenuates both agonist-stimulated production of nitric oxide and endothelium-dependent relaxation of isolated blood vessels. Our results indicate that Hsp90 facilitates signalling mediated by growth-factor, G-protein and mechanotransduction pathways that lead to the activation of eNOS. These observations indicate that in addition to its role as a molecular chaperone involved in protein folding and maturation, Hsp90 may also be recruited to cellular targets depending on the activation state of the cell.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Angiopoietin‐1 (Ang1) is an angiogenic growth factor that binds to the Tie2 receptor on vascular endothelium, promoting blood vessel maturation and integrity. In the present study, we have ...investigated whether Ang1 also possesses anti‐inflammatory properties by determining its effects on endothelial barrier function, neutrophil (PMN) adherence to endothelial cells (EC) and production of the PMN chemotactic factor interleukin‐8 (IL‐8).
Pretreatment of endothelial monolayers with Ang1 attenuated the permeability increase induced by thrombin in both lung microvascular cells and a human endothelial cell line. Similarly, Ang1 prevented the permeability‐inducing effects of platelet‐activating factor, bradykinin and histamine.
Pretreatment of EC with Ang1 also reduced the adherence of PMN to EC stimulated by thrombin. In contrast to its ability to counteract the increase in monolayer permeability brought about by various inflammatory agents, Ang1 did not affect the ability of histamine, PAF, or tumor necrosis factor‐α to stimulate PMN adherence to EC.
In addition to its ability to inhibit PMN adherence, Ang1 diminished IL‐8 production from EC challenged with thrombin in a concentration‐dependent manner.
When EC were preincubated with the specific Rho kinase (ROCK) inhibitor Y‐27632, we observed a reduction in PMN adherence in response to thrombin, as well as a decrease in thrombin‐stimulated IL‐8 production. Coincubation of monolayers with Y‐27632 and Ang1 did not further attenuate the above‐mentioned responses. However, Ang‐1 failed to inhibit the activation of RhoA in response to thrombin, suggesting that inhibition of EC adhesiveness for PMN and IL‐8 production by Ang1 does not result from reduced ROCK activation.
We conclude that Ang1 can counteract several aspects of the inflammatory response, including endothelial permeability, PMN adherence to EC as well as inhibition of IL‐8 production by EC.
British Journal of Pharmacology (2003) 139, 329–336. doi:10.1038/sj.bjp.0705259
Soluble guanylyl cyclase (sGC) is a cGMP-generating enzyme carrying a heme prosthetic group that functions as a nitric oxide (NO) sensor. sGC is present in most cells types, including the vascular ...endothelium, where its biological functions remain largely unexplored. Herein, we have investigated the role of sGC in angiogenesis and angiogenesis-related properties of endothelial cells (EC). Initially, we determined that sGC was present and enzymatically active in the chicken chorioallantoic membrane (CAM) during the days of maximal angiogenesis. In the CAM, inhibition of endogenous sGC inhibited neovascularization, whereas activation promoted neovessel formation. Using zebrafish as a model for vascular development, we did not detect any effect on vasculogenesis upon sGC blockade, but we did observe an abnormal angiogenic response involving the cranial and intersegmental vessels, as well as the posterior cardinal vein. In vitro, pharmacological activation of sGC or adenovirus-mediated sGC gene transfer promoted EC proliferation and migration, whereas sGC inhibition blocked tube-like network formation. In addition, sGC inhibition blocked the migratory response to vascular EC growth factor. Cells infected with sGC-expressing adenoviruses exhibited increased extracellular signal-regulated kinase 1/2 and p38 MAPK activation that was sensitive to sGC inhibition by 1H-1,2,4oxadiazolo4,3-aquinoxalin-1-one, suggesting that these mitogen-activated protein kinases are downstream effectors of sGC in EC. A functional role for p38 in cGMP-stimulated migration was demonstrated using SB203580 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; pharmacological inhibition of p38 attenuated BAY 41-2272 5-cyclopropyl-2-1-(2-fluoro-benzyl)-1H-pyrazolo3,4-bpyridin-3-yl-pyrimidin-4-ylamine and sGC overexpression-induced EC mobilization. We conclude that sGC activation promotes the expression of angiogenesis-related properties by EC and that sGC might represent a novel target to modulate neovessel formation.
In vascular tissues, hydrogen sulphide (H2S) is mainly produced from L‐cysteine by the cystathionine gamma‐lyase (CSE) enzyme. Recent studies show that administration of H2S to endothelial cells in ...culture stimulates cell proliferation, migration and tube formation. In addition, administration of H2S to chicken chorioallantoic membranes stimulates blood vessel growth and branching. Furthermore, in vivo administration of H2S to mice stimulates angiogenesis, as demonstrated in the Matrigel plug assay. Pathways involved in the angiogenic response of H2S include the PI‐3K/Akt pathway, the mitogen activated protein kinase pathway, as well as ATP‐sensitive potassium channels. Indirect evidence also suggests that the recently demonstrated role of H2S as an inhibitor of phosphodiesterases may play an additional role in its pro‐angiogenic effect. The endogenous role of H2S in the angiogenic response has been demonstrated in the chicken chorioallantoic membranes, in endothelial cells in vitro and ex vivo. Importantly, the pro‐angiogenic effect of vascular endothelial growth factor (but not of fibroblast growth factor) involves the endogenous production of H2S. The pro‐angiogenic effects of H2S are also apparent in vivo: in a model of hindlimb ischaemia‐induced angiogenesis, H2S induces a marked pro‐angiogenic response; similarly, in a model of coronary ischaemia, H2S exerts angiogenic effects. Angiogenesis is crucial in the early stage of wound healing. Accordingly, topical administration of H2S promotes wound healing, whereas genetic ablation of CSE attenuates it. Pharmacological modulation of H2S‐mediated angiogenic pathways may open the door for novel therapeutic approaches.
LINKED ARTICLES This article is part of a themed issue on Vascular Endothelium in Health and Disease. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.164.issue‐3
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