Abstract-Vascular dysfunction is a major complication of metabolic disorders such as diabetes and obesity. The current studies were undertaken to determine whether inflammatory responses are activated in the vasculature of mice with diet-induced obesity, and if so, whether Toll-Like Receptor-4 (TLR4), a key mediator of innate immunity, contributes to these responses. Mice lacking TLR4 (TLR4 Ϫ/Ϫ ) and wild-type (WT) controls were fed either a low fat (LF) control diet or a diet high in saturated fat (HF) for 8 weeks. In response to HF feeding, both genotypes displayed similar increases of body weight, body fat content, and serum insulin and free fatty acid (FFA) levels compared with mice on a LF diet. In lysates of thoracic aorta from WT mice maintained on a HF diet, markers of vascular inflammation both upstream (IKK␤ activity) and downstream of the transcriptional regulator, NF-B (ICAM protein and IL-6 mRNA expression), were increased and this effect was associated with cellular insulin resistance and impaired insulin stimulation of eNOS. In contrast, vascular inflammation and impaired insulin responsiveness were not evident in aortic samples taken from TLR4 Ϫ/Ϫ mice fed the same HF diet, despite comparable increases of body fat mass. Incubation of either aortic explants from WT mice or cultured human microvascular endothelial cells with the saturated FFA, palmitate (100 mol/L), similarly activated IKK␤, inhibited insulin signal transduction and blocked insulin-stimulated NO production. Each of these effects was subsequently shown to be dependent on both TLR4 and NF-B activation. These findings identify the TLR4 signaling pathway as a key mediator of the deleterious effects of palmitate on endothelial NO signaling, and are the first to document a key role for TLR4 in the mechanism whereby diet-induced obesity induces vascular inflammation and insulin resistance.

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Many of the proinflammatory effects of gram-negative bacteria are elicited by the interaction of bacterial lipopolysaccharide (LPS) with Toll-like receptor 4 (TLR4) expressed on host cells. TLR4 signaling leads to activation of NF-κB and transcription of many genes involved in the inflammatory response. In this study, we examined the signaling pathways involved in NF-κB activation by TLR4 signaling in human microvascular endothelial cells. Akt is a major downstream target of phosphoinositide 3 kinase (PI3-kinase), and PI3-kinase activation is necessary and sufficient for Akt phosphorylation. Consequently, Akt kinase activation was used as a measure of PI3-kinase activity. In a stable transfection system, dominant-negative mutants of myeloid differentiation factor 88 (MyD88) and interleukin-1 (IL-1) receptor-associated kinase 1 (IRAK-1) (MyD88-TIR and IRAK-DD, respectively) blocked Akt kinase activity in response to LPS and IL-1β. A dominant-negative mutant (Mal-P/H) of MyD88 adapter-like protein (Mal), a protein with homology to MyD88, failed to inhibit LPS- or IL-1β-induced Akt activity. Moreover, a dominant-negative mutant of p85 (p85-DN) inhibited the NF-κB luciferase activity, IL-6 production, and IκBα degradation elicited by LPS and IL-1β but not that stimulated by tumor necrosis factor alpha. The dominant-negative mutant of Akt partially inhibited the NF-κB luciferase activity evoked by LPS and IL-1β. However, expression of a constitutively activated Akt failed to induce NF-κB luciferase activity. These findings indicate that TLR4- and IL-1R-induced PI3-kinase activity is mediated by the adapter proteins MyD88 and IRAK-1 but not Mal. Further, these studies suggest that PI3-kinase is an important mediator of LPS and IL-1β signaling leading to NF-κB activation in endothelial cells and that Akt is necessary but not sufficient for NF-κB activation by TLR4.

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Transgenic mice that over-express B cell leukemia/lymphomas (Bcl)-2 in myeloid cells under control of the human MRP8 promoter (hMRP8-Bcl-2) or in T lymphocytes under the Eμ promoter (Eμ-Bcl-2) were compared with C57BL/6 control mice following cecal ligation and puncture (CLP). There was a significant difference in outcome between the hMRP8-Bcl-2 and control mice with 100% survival in the hMRP8-Bcl-2 mice vs 25% survival in the control mice. In separate experiments there was a significant difference between Eμ-Bcl-2 and control mice with 87.5 and 22.2% survival, respectively. Adoptive transfer of CD11b-positive bone marrow cells from hMRP8-Bcl-2 or C57BL/6 mice to C57BL/6 mice subjected to CLP resulted in 100 and 0% survival, respectively. Adoptive transfer of CD11b-positive cells from either hMRP8-Bcl-2 or C57BL/6 mice to Rag-1−/− mice (no mature T or B cells) subjected to CLP resulted in survival of 87.5 and 12.5%, respectively. The hMRP8-Bcl-2 mice had significantly more neutrophils and fewer bacteria in the peritoneum compared with C57BL/6 mice 24 h after CLP. These experiments show that Bcl-2 over-expression is protective in CLP and that protection is independent of lymphocytes. We propose that over-expression of Bcl-2 in T cells or myeloid cells induce release of a molecule(s) that protects against death following CLP.

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A novel, serum albumin-binding glycoprotein of molecular weight (mw) 43,000 (43K protein) was initially purified from the culture medium of bovine aortic endothelial (BAE) cells (Sage, H., Johnson, C., and Bornstein, P., J. Biol. Chem. 259:3993-4007, 1984). Its secretion by normal mesenchymal cells and by transformed cells of both ectodermal and endodermal origin suggested a general role in cellular function. To examine the effect of sublethal injury in vitro on the biosynthesis of 43K protein, BAE cells were exposed to endotoxin. At concentrations which produced minimal cell detachment and lysis, the cells secreted 70-100% more protein compared to control cultures, and the relative increase in 43K protein over total protein was approximately three-fold. A second type of cellular injury, manifested by rapid cellular proliferation and migration in response to sparse plating density (a condition that we have termed 'culture shock'), was also accompanied by a significant increase in the secretion of 43K protein. Pulse-chase studies revealed that the initial product secreted within 1.5 h was of Mr 38,000, and that between 6 and 21 h this molecule was converted to the final form of Mr 43,000. The 43K protein was not associated with RNA or glycosaminoglycan, but appeared to be linked to complex oligosaccharides containing peripheral sialosyl residues. Treatment with tunicamycin produced lower mw forms that displayed reduced affinity for albumin. By immunologic criteria, peptide mapping, and amino acid analysis, the 43K protein was shown to be structurally distinct from several proteins of Mr 40,000-50,000 associated with endothelium or with serum, including tissue factor, a plasminogen anti-activator, and several apolipoproteins. In addition, the 43K protein was not present in the extracellular matrices of endothelial, fibroblastic, or smooth muscle cells, nor was it found in plasma, serum, platelet releasate, or alveolar lavage fluids. These studies identify a unique Mr 43,000 glycoprotein that is associated with cellular stress or injury in vitro. As a secreted but nonmatrix macromolecule, this protein may be part of a 'survival kit' used by the endothelium to cope with cellular injury.

The vascular endothelium is a key target of circulating bacterial lipopolysaccharide (LPS). LPS elicits a wide array of endothelial responses, including the upregulation of cytokines, adhesion molecules, and tissue factor, many of which are dependent on NF-B activation. In addition, LPS has been demonstrated to induce endothelial apoptosis both in vitro and in vivo. Although the mechanism by which LPS activates NF-B has been well elucidated, the signaling pathway(s) involved in LPS-induced apoptosis remains unknown. Using a variety of dominant negative constructs, we have identified a role for MyD88 and interleukin-1 receptor-associated kinase-1 (IRAK-1) in mediating LPS pro-apoptotic signaling in human endothelial cells. We also demonstrate that LPS-induced endothelial NF-B activation and apoptosis occur independent of one another. Together, these data suggest that the proximal signaling molecules involved in LPS-induced NF-B activation have a requisite involvement in LPS-induced apoptosis and that the pathways leading to NF-B activation and apoptosis diverge downstream of IRAK-1.Gram-negative bacterial sepsis is a life-threatening event that afflicts ϳ200,000 patients annually in the United States (1). A deleterious outcome of sepsis is the development of septic shock. Endothelial cell (EC) 1 injury and/or dysfunction is a commonality among several key complications associated with septic shock, including systemic vascular collapse, disseminated intravascular coagulation, and vascular leak syndromes (2, 3). The pathogenesis of septic shock and its attendant vascular complications is believed to be mediated, at least in part, by lipopolysaccharide (LPS), a component of the outer envelope of all Gram-negative bacteria (4 -6).LPS directly activates the vascular endothelium and elicits an array of EC responses including the up-regulation of proinflammatory cytokines, adhesion molecules, and tissue factor. Many of these responses are mediated by NF-B (7). The signaling pathway by which LPS activates NF-B in EC has only recently been elucidated (8). Circulating LPS binds to the acute phase protein, LPS-binding protein (LBP), and this complex is recognized by soluble CD-14 (sCD14) (9). Although the exact mechanism of interaction remains unknown, this LPS⅐LPS-binding protein⅐soluble CD14 complex is recognized by the transmembrane receptor, Toll-like receptor (Tlr)-4 (10). The extracellular domain of Tlr-4 contains repeating leucine-rich motifs, and the cytoplasmic portion contains a Toll receptorinterleukin-1 receptor (TIR) domain with homology to the intracellular signaling domain of the type 1 interleukin-1 receptor (11). After Tlr-4 activation, another TIR domain-containing protein, MyD88, is recruited to Tlr-4 through respective TIR-TIR interactions (12, 13). MyD88 also contains a death domain (DD), a highly conserved protein binding domain that facilitates its interaction with another DD-containing signaling molecule, interleukin-1 receptor-associated kinase-1 (IRAK-1) (11). IRAK-1 subsequently autophosphorylates...