Background-The inability to inhibit multiple mediators of septic shock represents a major hurdle in the treatment of septic shock. In vivo inhibition of nuclear factor (NF)-kappaB activation, a transcription factor regulating expression of many proinflammatory genes, could provide a useful strategy for the treatment of septic shock. Methods and Results-In rats challenged with lipopolysaccharide (LPS) 8 mg/kg IV, we determined the time course of NF-kappaB activation and expression of multiple inflammatory signals: tumor necrosis factor-alpha (TNF-alpha), cyclooxygenase-2 (COX-2), cytokine-inducible neutrophil chemoattractant (CINC), and intercellular adhesion molecule-1 (ICAM)-1. We studied the effects of in vivo inhibition of NF-kappaB activation using pyrrolidine dithiocarbamate (PDTC) on the expression of these mediators. NF-kappaB activation preceded the induction of TNF-alpha, COX-2, CINC, and ICAM-1 mRNAs. PDTC prevented the LPS-induced NF-kappaB activation but did not inhibit activation of the transcription factors AP-1, Sp-1, and CREB. PDTC inhibited the LPS-induced expression of TNF-alpha, COX-2, CINC, and ICAM-1 mRNA and proteins and reduced the LPS-induced increases in plasma TNF-alpha, 6-keto-prostaglandin F(1alpha), and CINC concentrations. Inhibition of expression of these mediators prevented the increases in myeloperoxidase activity (a measure of neutrophil sequestration) in the heart, lungs, and liver. Conclusions-NF-kappaB activation correlates with LPS-induced expression of TNF-alpha, COX-2, CINC, and ICAM-1 genes in vivo. PDTC inhibits NF-kappaB activation and expression of these proinflammatory genes and their products. Thus, blocking NF-kappaB activation may be an effective strategy in the treatment of septic shock.

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To defi ne the roles of endothelial-intrinsic nuclear factor B (NF-B) activity in host defense and multiple organ injury in response to sepsis, we generated double transgenic (TG) mice (EC-rtTA/I-B ␣ mt) that conditionally overexpress a degradation-resistant form of the NF-B inhibitor I-B ␣ (I-B ␣ mt) selectively on vascular endothelium. The ECrtTA/I-B ␣ mt mice had no basal, but a relatively high level of doxycycline-inducible, I-B ␣ mt expression. I-B ␣ mt expression was detected in endothelial cells, but not in fi broblasts, macrophages, and whole blood cells, confi rming that transgene expression was restricted to the endothelium. When subjected to endotoxemia, EC-rtTA/I-B ␣ mt mice showed endothelial-selective blockade of NF-B activation, repressed expression of multiple endothelial adhesion molecules, reduced neutrophil infi ltration into multiple organs, decreased endothelial permeability, ameliorated multiple organ injury, reduced systemic hypotension, and abrogated intravascular coagulation. When subjected to cecal ligation and puncture -induced sepsis, the TG mice had less severe multiple organ injury and improved survival compared with wild-type (WT) mice. WT and EC-rtTA/I-B ␣ mt mice had comparable capacity to clear three different pathogenic bacteria. Our data demonstrate that endothelial NF-B activity is an essential mediator of septic multiple organ infl ammation and injury but plays little role in the host defense response to eradicate invading pathogenic bacteria.

Although the role of systemic activation of the nuclear factor kappaB (NF-kappaB) pathway in septic coagulation has been well documented, little is known about the contribution of endothelial-specific NF-kappaB signaling in this pathologic process. Here, we used transgenic mice that conditionally overexpress a mutant I-kappaBalpha, an inhibitor of NF-kappaB, selectively on endothelium, and their wild-type littermates to define the role of endothelial-specific NF-kappaB in septic coagulation. In wild-type mice, lipopolysaccharide (LPS) challenge (5 mg/kg intraperitoneally) caused markedly increased plasma markers of coagulation, decreased plasma fibrinogen level, and widespread tissue fibrin deposition, which were abrogated by endothelial NF-kappaB blockade in transgenic mice. Endothelial NF-kappaB blockade inhibited tissue factor expression in endothelial cells, but not in leukocytes. Endothelial NF-kappaB blockade did not inhibit LPS-induced tissue factor expression in heart, kidney, and liver. Endothelial NF-kappaB blockade prevented LPS down-regulation of endothelial protein C receptor (EPCR) and thrombomodulin protein expressions, inhibited tissue tumor necrosis factor-alpha converting enzyme activity, reduced EPCR shedding, and restored plasma protein C level. Our data demonstrate that endothelial intrinsic NF-kappaB signaling plays a pivotal role in septic coagulation and suggests a link between endothelial-specific NF-kappaB activation and the impairment of the thrombomodulin-protein C-EPCR anticoagulation pathway.

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Although the role of NF-κB in the pathogenesis of sepsis and septic shock has been extensively studied, little is known about the causative contribution of endothelial-intrinsic NF-κB to these pathological processes. In this study, we used transgenic (TG) mice (on FVB genetic background) that conditionally overexpress the NF-κB inhibitor, mutant I-κBα, selectively on endothelium and their transgene-negative littermates (wild type (WT)) to define the causative role of endothelial-specific NF-κB signaling in septic shock and septic vascular dysfunction. In WT mice, LPS challenge caused systemic hypotension, a significantly blunted vasoconstrictor response to norepinephrine, and an impaired endothelium-dependent vasodilator response to acetylcholine, concomitant with a markedly increased aortic inducible NO synthase expression, significantly elevated plasma and aortic levels of nitrite/nitrate, increased aortic TNF-α expression, and decreased aortic endothelial NO synthase (eNOS) expression. In TG mice whose endothelial NF-κB was selectively blocked, LPS caused significantly less hypotension and no impairments in vasoconstrictor and endothelium-dependent vasodilator responses, associated with significantly reduced aortic inducible NO synthase expression, decreased plasma and aortic levels of nitrite/nitrate, reduced aortic TNF-α expression, and increased aortic eNOS expression. TNF-α knockout mice prevented LPS-induced eNOS down-regulation. WT mice subjected to cecal ligation and puncture showed significant systemic hypotension, which was prevented in TG mice. Our data show that selective blockade of endothelial-intrinsic NF-κB pathway is sufficient to abrogate the cascades of molecular events that lead to septic shock and septic vascular dysfunction, demonstrating a pivotal role of endothelial-specific NF-κB signaling in the pathogenesis of septic shock and septic vascular dysfunction.

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An increasing body of evidence suggests that ribosomal proteins may have ribosome-independent functions and may be involved in various physiological and pathological processes. To examine the role of ribosomal protein L34 (RPL34) in cancer transformation, we assessed its expression in gastric cancer cell lines and found it highly expressed. We further used lentivirus-mediated small interfering RNAs (siRNAs) to knockdown RPL34 expression in the human gastric cancer cell line SGC-7901. RNA interference (RNAi)-mediated inhibition of RPL34 expression in SGC-7901 cells significantly suppressed cell proliferation, increased apoptosis and arrested cells in the S phase. The results of the present study suggest that RPL34 plays a critical role in cell proliferation, cell cycle distribution and apoptosis of human malignant gastric cells.