Endothelial protease-activated receptor-2 induces tissue factor expression and von Willebrand factor release

A second protease-activated receptor (PAR-2) that could be activated by trypsin or more physiologically by mast cell tryptase has been recently cloned. Both the structure and activation mechanism of PAR-2 was similar to the functional thrombin receptor (PAR-1). Although many effects of the coagulation protease thrombin on the vascular endothelium could be attributed to PAR-1 activation, very little is known about the physiological and pathophysiological role of PAR-2. We investigated whether stimulation of PAR-2 on endothelial cells induced two cellular responses that play a central role in primary and secondary haemostasis: the release of high molecular weight von Willebrand factor (hmw-VWF) from Weibel-Palade bodies and the de novo synthesis of tissue factor (TF) mRNA and protein. Human umbilical vein endothelial cells (HUVEC) were incubated with agonists for PAR-2 at 37 degrees C. Both trypsin and SLIGKV increased TF mRNA and activity and induced the release of hmw-VWF due to elevated levels of cytosolic Ca2+. Trypsin (10 nm) induced a 6-fold increase of TF mRNA and reduced time until fibrin clot formation to 37%, indicating trebling of the cell surface located TF activity. Stimulation of HUVEC with the PAR-2 agonist peptide SLIGKV induced a dose-dependent increase of TF mRNA up to 6 times and TF activity up to 3 times. Release of hmw-VWF was achieved both after incubation of HUVEC with trypsin and SLIGKV and was directly depending on intracellular Ca2+ mobilization. To make results comparable to the functional thrombin receptor, homologous experiments were carried out using the PAR-1 agonists thrombin and SFLLRN.     

Atheroprotection via cannabinoid receptor-2 is mediated by circulating and vascular cells in vivo

Low-dose oral tetrahydrocannabinol (THC) reduces progression of atherosclerosis in mice. THC activates central cannabinoid-1 receptors (CB1) with subsequent psychoactive effects as well as peripheral cannabinoid-2 receptors (CB2). In order to dissect the underlying mechanisms, we performed experiments under selective CB2 stimulation as well as after genetic disruption of the CB2 receptor. Atherosclerosis prone apolipoprotein E-deficient mice were crossed with cannabinoid receptor-2 deficient mice to obtain ApoE −/− CB2 −/− double knockout mice. After 8 weeks of a high-cholesterol diet, immunohistochemical stainings of the aortic root revealed that vascular leukocyte infiltration in atherosclerotic plaques was accelerated in ApoE −/− CB2 −/− mice compared with ApoE −/− mice. This was accompanied by increased release of reactive oxygen species as measured using L012-enhanced chemiluminescence, and by decreased endothelial function as assessed in isolated aortic rings in organ chamber experiments. ApoE −/− mice treated with the selective CB2 agonist JWH 133 during a high-cholesterol diet showed decreased atherosclerotic lesion formation, improved endothelial function and reduced levels of reactive oxygen species. To assess whether CB2 expression in circulating cells influences atherosclerosis, irradiated ApoE −/− mice were repopulated with bone marrow-derived cells from ApoE −/− and ApoE −/− CB2 −/− mice and were fed a high-cholesterol diet for 8 weeks. CB2 deficiency in bone marrow-derived cells increased leukocyte infiltration into the vessel wall, but had no impact on plaque formation. Cell culture experiments revealed that CB2 activation diminishes ROS generation in vascular cells. Selective CB2 receptor stimulation modulates atherogenesis via impact on both circulating proinflammatory and vascular cells.     

Atheroprotection via cannabinoid receptor-2 is mediated by circulating and vascular cells in vivo

Low-dose oral tetrahydrocannabinol (THC) reduces progression of atherosclerosis in mice. THC activates central cannabinoid-1 receptors (CB1) with subsequent psychoactive effects as well as peripheral cannabinoid-2 receptors (CB2). In order to dissect the underlying mechanisms, we performed experiments under selective CB2 stimulation as well as after genetic disruption of the CB2 receptor. Atherosclerosis prone apolipoprotein E-deficient mice were crossed with cannabinoid receptor-2 deficient mice to obtain ApoE -/- CB2 -/- double knockout mice. After 8weeks of a high-cholesterol diet, immunohistochemical stainings of the aortic root revealed that vascular leukocyte infiltration in atherosclerotic plaques was accelerated in ApoE -/- CB2 -/- mice compared with ApoE -/- mice. This was accompanied by increased release of reactive oxygen species as measured using L012-enhanced chemiluminescence, and by decreased endothelial function as assessed in isolated aortic rings in organ chamber experiments. ApoE -/- mice treated with the selective CB2 agonist JWH 133 during a high-cholesterol diet showed decreased atherosclerotic lesion formation, improved endothelial function and reduced levels of reactive oxygen species. To assess whether CB2 expression in circulating cells influences atherosclerosis, irradiated ApoE -/- mice were repopulated with bone marrow-derived cells from ApoE -/- and ApoE -/- CB2 -/- mice and were fed a high-cholesterol diet for 8weeks. CB2 deficiency in bone marrow-derived cells increased leukocyte infiltration into the vessel wall, but had no impact on plaque formation. Cell culture experiments revealed that CB2 activation diminishes ROS generation in vascular cells. Selective CB2 receptor stimulation modulates atherogenesis via impact on both circulating proinflammatory and vascular cells.     

Toll-like receptor-2 mediates mycobacteria-induced proinflammatory signaling in macrophages

The recognition of mycobacterial cell wall components causes macrophages to secrete tumor necrosis factor alpha (TNF-alpha) and other cytokines that are essential for the development of a protective inflammatory response. We show that toll-like receptors are required for the induction of TNF-alpha in macrophages by Mycobacterium tuberculosis. Expression of a dominant negative form of MyD88 (a signaling component required for toll-like receptor signaling) in a mouse macrophage cell line blocks TNF-alpha production induced by M. tuberculosis. We identify toll-like receptor-2 (TLR2) as the specific toll-like receptor required for this induction by showing that expression of an inhibitory TLR2 (TLR2-P681H) blocks TNF-alpha production induced by whole M. tuberculosis. Further, we show that TLR2-dependent signaling mediates responses to mycobacterial cell wall fractions enriched for lipoarrabinomannan, mycolylarabinogalactan-peptidoglycan complex, or M. tuberculosis total lipids. Thus, although many mycobacterial cell wall fractions are identified to be inflammatory, all require TLR2 for induction of TNF-alpha in macrophages. These data suggest that TLR2 is essential for the induction of a protective immune response to mycobacteria.     

VEGF treatment induces signaling pathways that regulate both actin polymerization and depolymerization

The angiogenic growth factor vascular endothelial growth factor (VEGF) enhances endothelial cell migration through the activation of multiple signaling transduction pathways. Actin reorganization is an important component in VEGF-induced migration, yet the signaling pathways mediating this process remain unclear. Actin reorganization involves both actin polymerization and depolymerization, and in this study we demonstrate that VEGF-treatment regulates both of these activities. With respect to actin polymerization, our results indicate that the actin nucleation promoting factors (NPF) neural Wiskott–Aldrich syndrome protein (N-WASP) binds the SH2- plus SH3-domain containing adaptor protein Nck in both control and VEGF-treated cells. We had previously showed that VEGF treatment leads to the recruitment of Nck to activated receptor, and our current results indicate a VEGF-dependent redistribution of N-WASP to the cell surface. A Nck dominant-negative blocked Nck recruitment to receptor, blocked N-WASP cellular redistribution and attenuated actin stress fiber formation. With respect to actin depolymerization, VEGF-treatment led to the rapid phosphorylation of the actin depolymerization factor cofilin, and its upstream regulator, LIM-kinase (LIMK). Unlike what is observed in certain other cell types, the p21-activated kinase (PAK), a Nck binding protein, does not mediate VEGF-induced LIMK phosphorylation, as a PAK dominant-negative had no effect on this activity. The PAK dominant-negative also did not affect VEGF-induced actin reorganization. Pharmacological inhibitors of phosphoinositide-3 kinase (PI3-K) and the rho-activated kinase (ROCK) attenuated VEGF-induced LIMK phosphorylation, indicating a role for (PI3-K) and ROCK in the signaling pathways leading to regulation of LIMK activity.     

Hypoglycaemia induces decreased islet blood perfusion mediated by the central nervous system in normal and Type 2 diabetic GK rats

Aims/hypothesis The aim of this study was to evaluate the effects of induced hypoglycaemia on pancreatic-islet blood flow in normal rats and in the GK rat, an animal model of Type 2 diabetes which normally has an increased islet blood perfusion.Methods A 50% reduction in blood glucose concentrations was achieved by intravenous administration of a rapidly acting insulin (15 IU/kg body weight). Blood flows were measured by a non-radioactive microsphere technique.Results A pronounced decrease in islet blood flow was observed in all animals, but preferentially in the Type 2 diabetic GK rats. When a similar dose of insulin was given to whole-pancreas transplanted rats only islet blood flow in the native pancreas was decreased, whereas that of the transplanted, i.e. denervated, pancreas was unchanged. Administration of 2-deoxy-D-glucose, which induces intracellular glucopenia especially in neurons, also decreased islet blood flow despite a systemic hyperglycaemia.Conclusion/interpretation Hypoglycaemia leads to a preferential decrease in pancreatic-islet blood perfusion. The effect is probably mediated by the central nervous system, since 2-deoxy D-glucose-induced neuronal glucopenia caused a similar decrease in blood flow. The effects of islet blood flow are not likely to be mediated by nervous stimulation of the adrenal glands, with an associated release of catecholamines, because the transplanted pancreas was not affected by hypoglycaemia. The decreased islet blood perfusion could possibly diminish the output of insulin from the islets, thereby preventing a further decrease in blood glucose concentrations.Abbreviations GK Goto-Kakizaki     

Rapamycin induces tumor-specific thrombosis via tissue factor in the presence of VEGF

Therapeutic strategies that target and disrupt the already-formed vessel networks of growing tumors are actively pursued. The goal of these approaches is to induce a rapid shutdown of the vascular function of the tumor so that blood flow is arrested and tumor cell death occurs. Here we show that the mammalian target of rapamycin (mTOR) inhibitor rapamycin, when administered to tumor-bearing mice, selectively induced extensive local microthrombosis of the tumor microvasculature. Importantly, rapamycin administration had no detectable effect on the peritumoral or normal tissue. Intravital microscopy analysis of tumors implanted into skinfold chambers revealed that rapamycin led to a specific shutdown of initially patent tumor vessels. In human umbilical vein endothelial cells vascular endothelial growth factor (VEGF)-induced tissue factor expression was strongly enhanced by rapamycin. We further show by Western blot analysis that rapamycin interferes with a negative feedback mechanism controlling this pathologic VEGF-mediated tissue factor expression. This thrombogenic alteration of the endothelial cells was confirmed in a one-step coagulation assay. The circumstance that VEGF is up-regulated in most tumors may explain the remarkable selectivity of tumor vessel thrombosis under rapamycin therapy. Taken together, these data suggest that rapamycin, besides its known antiangiogenic properties, has a strong tumor-specific, antivascular effect in tumors.     

Modulating Toll-like receptor mediated signaling by (1-->3)-beta-D-glucan rapidly induces cardioprotection

OBJECTIVE: Immune and inflammatory signaling pathways, initiated by the innate response, are involved in myocardial ischemia/reperfusion (I/R) injury. Toll-like receptor (TLR) mediated MyD88-dependent NFkappaB pathways play a role in the induction of innate immunity. We have reported that glucan phosphate (GP) improved survival in experimental sepsis, which correlated with decreased tissue NFkappaB activation. In the present study, we report that GP rapidly induced cardioprotection against I/R injury in vivo. METHODS: Sprague-Dawley rats were pretreated with GP (40 mg/kg, i.p) 1 h before 45 min of ligation of the left anterior descending coronary followed by reperfusion for 4 and 24 h. Infarction size was examined by triphenyltetrazolium chloride (TTC) staining. NFkappaB activation was analyzed by electrophoretic mobility shift assay (EMSA). IkappaB kinase-beta (IKKbeta), IL-1 receptor-associated kinase (IRAK) and Phosphoinositide 3-kinase (PI3K) activities were determined by kinase assay with appropriate substrates. Association of TLR4 with MyD88 or with PI3K p85 was assessed by immunoprecipitation with anti-TLR4 followed by immunoblotting with anti-MyD88 or anti-p85. RESULTS: GP treatment reduced infarct size by 47% in rat hearts subjected to reperfusion for 4 h and by 50% following reperfusion for 24 h. The same protective effect was observed when GP was administrated 5 min after initiation of ischemia. The mechanisms of GP induced cardioprotection involve decreased association of TLR4 with MyD88, inhibition of I/R induced IRAK and IKKbeta activity and decreased NFkappaB activity. In addition, GP increased TLR4 phosphotyrosine, resulting in increasing PI3K/Akt activity in the myocardium, which correlated with decreased cardiac myocyte apoptosis following I/R. CONCLUSION: The results suggest that activation of the TLR mediated MyD88-dependent NFkappaB signaling pathway may play an important role in myocardial I/R injury, while stimulation of the PI3K/Akt signaling could serve a protective role. The data indicates that GP treatment shifts the TLR mediated activation signal in I/R from a predominantly NFkappaB pathway to a predominant PI3K/Akt signaling pathway.     

Cardiovascular phenotypes of kinin B2 receptor- and tissue kallikrein-deficient mice

To clarify the role of the kallikrein-kinin system in cardiovascular homeostasis, the systemic and regional hemodynamics of kinin B2 receptor-deficient (B2-/-) and tissue kallikrein-deficient (TK-/-) mice were compared with their wild-type (WT) littermates on a pure C57BL/6 genetic background. B2-/-, TK-/-, and WT adult mice were normotensive and displayed normal hemodynamic (left ventricular [LV] pressure, cardiac output, total peripheral resistance, dP/dt(max)) and echocardiographic (septum and LV posterior wall thickness, LV diameter, LV mass, and LV fractional shortening) parameters. However, heart rate was lower in B2-/- mice compared with TK-/- and WT mice. In addition, B2-/- mice, but not TK-/- mice, exhibited lower coronary and renal blood flows and greater corresponding vascular resistances than did WT mice, indicating a tonic physiological vasodilating effect of bradykinin in these vascular beds. However, maximal coronary vasodilatation capacity, estimated after dipyridamole infusion, was similar in the 3 groups of mice. B2-/- mice were significantly more sensitive than were TK-/- mice to the vasoconstrictor effects of angiotensin II and norepinephrine. Finally, renin mRNA levels were significantly greater in B2-/- mice and smaller in TK-/- mice compared with WT mice. Taken together, these results indicate that under basal conditions, the kinin B2 receptor is not an important determinant of blood pressure in mice but is involved in the control of regional vascular tone in the coronaries and the kidneys. The phenotypic differences observed between TK-/- and B2-/- mice could be underlain by tissue kallikrein kinin-independent effect and/or kinin B1 receptor activation.     

Proteinase-activated receptor-1 is an anti-inflammatory signal for colitis mediated by a type 2 immune response

BACKGROUND: Activation of colonic proteinase activated receptor-1 (PAR1) provokes colonic inflammation and increases mucosal permeability in mice. The mechanism of inflammation is not neurogenic like in the paw of rats but depends on PAR1-mediated activation monocytic cells. PAR1 activation in the colon increases the release of lymphocyte T helper-1 (TH1) cytokines. Moreover, PAR1 expression is increased in biopsies from patients with inflammatory bowel disease, and its activation during TH1-mediated colitis in mice increases all of the hallmarks of inflammation. METHODS: This study aimed to characterize the effects of PAR1 activation in oxazolone-mediated colitis, involving a TH2 cytokine profile. RESULTS: Intracolonic administration of oxazolone increased myeloperoxidase activity, damage score, and interleukin (IL)-4, IL-10, tumor necrosis factor alpha, and IL-1beta mRNA expression but lowered interferon-gamma mRNA expression, indicating colonic inflammation of a TH2 profile. The concurrent intracolonic administration of a PAR1 agonist in oxazolone-treated mice inhibited colitis, resulting in a reduction of myeloperoxidase activity, damage score, and inflammatory cytokine mRNA expression. Using PAR1-deficient mice, we confirmed that the anti-inflammatory effects of PAR1 agonists were mediated by PAR1. Moreover, in PAR1-deficient mice or in mice treated with a PAR1 antagonist, oxazolone-induced colitis was exacerbated, showing an endogenous modulatory role for PAR1 in this TH2 cytokine profile of colitis. CONCLUSIONS: Thus, as opposed to a previously shown proinflammatory role for PAR1 in a TH1 cytokine-mediated colitis, our new data show anti-inflammatory role for PAR1 activation in the setting of TH2 cytokine colitis model.