Bacterial LPS induces rapid thrombocytopenia, hypotension, and sepsis. Although growing evidence suggests that platelet activation plays a critical role in LPS-induced thrombocytopenia and tissue damage, the mechanism of LPS-mediated platelet activation is unclear. In this study, we show that LPS stimulates platelet secretion of dense and α granules as indicated by ATP release and P-selectin expression, and thus enhances platelet activation induced by low concentrations of platelet agonists. Platelets express components of the LPS receptor-signaling complex, including TLR (TLR4), CD14, MD2, and MyD88, and the effect of LPS on platelet activation was abolished by an anti-TLR4-blocking Ab or TLR4 knockout, suggesting that the effect of LPS on platelet aggregation requires the TLR4 pathway. Furthermore, LPS-potentiated thrombin- and collagen-induced platelet aggregation and FeCl3-induced thrombus formation were abolished in MyD88 knockout mice. LPS also induced cGMP elevation and the stimulatory effect of LPS on platelet aggregation was abolished by inhibitors of NO synthase and the cGMP-dependent protein kinase (PKG). LPS-induced cGMP elevation was inhibited by an anti-TLR4 Ab or by TLR4 deficiency, suggesting that activation of the cGMP/protein kinase G pathway by LPS involves the TLR4 pathway. Taken together, our data indicate that LPS stimulates platelet secretion and potentiates platelet aggregation through a TLR4/MyD88- and cGMP/PKG-dependent pathway.

Background: While tumour necrosis factor a (TNF-a) appears to be associated with the development of non-alcoholic steatohepatitis (NASH), its precise role in the pathogenesis of NASH is not well understood. Methods: Male mice deficient in both TNF receptors 1 (TNFR1) and 2 (TNFR2) (TNFRDKO mice) and wildtype mice were fed a methionine and choline deficient (MCD) diet or a control diet for eight weeks, maintaining isoenergetic intake. Results: MCD dietary feeding of TNFRDKO mice for eight weeks resulted in attenuated liver steatosis and fibrosis compared with control wild-type mice. In the liver, the number of activated hepatic Kupffer cells recruited was significantly decreased in TNFRDKO mice after MCD dietary feeding. In addition, hepatic induction of TNF-a, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 was significantly suppressed in TNFRDKO mice. While in control animals MCD dietary feeding dramatically increased mRNA expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) in both whole liver and hepatic stellate cells, concomitant with enhanced activation of hepatic stellate cells, both factors were significantly lower in TNFRDKO mice. In primary cultures, TNF-a administration enhanced TIMP-1 mRNA expression in activated hepatic stellate cells and suppressed apoptotic induction in activated hepatic stellate cells. Inhibition of TNF induced TIMP-1 upregulation by TIMP-1 specific siRNA reversed the apoptotic suppression seen in hepatic stellate cells. Conclusions: Enhancement of the TNF-a/TNFR mediated signalling pathway via activation of Kupffer cells in an autocrine or paracrine manner may be critically involved in the pathogenesis of liver fibrosis in this NASH animal model.

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Elevated generation of reactive oxygen species (ROS) by endothelial enzymes, including NADPH-oxidase, is implicated in vascular oxidative stress and endothelial proinflammatory activation involving exposure of vascular cell adhesion molecule-1 (VCAM-1). Catalase and superoxide dismutase (SOD) conjugated with antibodies to platelet/endothelial cell adhesion molecule 1 (PECAM-1) bind specifically to endothelium and inhibit effects of corresponding ROS, H(2)O(2), and superoxide anion. In this study, anti-PECAM/SOD, but not anti-PECAM/catalase or nontargeted enzymes, including polyethylene glycol (PEG)-SOD, inhibited 2- to 3-fold VCAM expression caused by tumor necrosis factor (TNF), interleukin-1β, and lipopolysaccharide. Anti- PECAM/SOD, but not nontargeted counterparts, accumulated in vascular endothelium after intravenous injection, localized in endothelial endosomes, and inhibited by 70% lipopolysaccharide-caused VCAM-1 expression in mice. Anti-PECAM/SOD colocalized with EEA-1-positive endothelial vesicles and quenched ROS produced in response to TNF. Inhibitors of NADPH oxidase and anion channel ClC3 blocked TNF-induced VCAM expression, affirming that superoxide produced and transported by these proteins, respectively, mediates inflammatory signaling. Anti-PECAM/SOD abolished VCAM expression caused by poly(I:C)-induced activation of toll-like receptor 3 localized in intracellular vesicles. These results directly implicate endosomal influx of superoxide in endothelial inflammatory response and suggest that site-specific interception of this signal attained by targeted delivery of anti-PECAM/SOD into endothelial endosomes may have anti-inflammatory effects.