Identification of plumericin as a potent new inhibitor of the NF-κB pathway with anti-inflammatory activity in vitro and in vivo.

BACKGROUND AND PURPOSE: The transcription factor NF-κB orchestrates many pro-inflammatory signals and its inhibition is considered a promising strategy to combat inflammation. Here we report the characterization of the natural product plumericin as a highly potent inhibitor of the NF-κB pathway with a novel chemical scaffold, which was isolated via a bioactivity-guided approach, from extracts of Himatanthus sucuuba, an Amazonian plant traditionally used to treat inflammation-related disorders. EXPERIMENTAL APPROACH: A NF-κB luciferase reporter gene assay was used to identify NF-κB pathway inhibitors from H. sucuuba extracts. Monitoring of TNF-α-induced expression of the adhesion molecules VCAM-1, ICAM-1 and E-selectin by flow cytometry was used to confirm NF-κB inhibition in endothelial cells, and thioglycollate-induced peritonitis in mice to confirm effects in vivo. Western blotting and transfection experiments were used to investigate the mechanism of action of plumericin. KEY RESULTS: Plumericin inhibited NF-κB-mediated transactivation of a luciferase reporter gene (IC50 1 µM), abolished TNF-α-induced expression of the adhesion molecules VCAM-1, ICAM-1 and E-selectin in endothelial cells and suppressed thioglycollate-induced peritonitis in mice. Plumericin exerted its NF-κB pathway inhibitory effect by blocking IκB phosphorylation and degradation. Plumericin also inhibited NF-κB activation induced by transfection with the constitutively active catalytic subunit of the IκB kinase (IKK-ß), suggesting IKK involvement in the inhibitory action of this natural product. CONCLUSION AND IMPLICATIONS: Plumericin is a potent inhibitor of NF-κB pathways with a new chemical scaffold. It could be further explored as a novel anti-inflammatory lead compound.

Cold induces reactive oxygen species production and activation of the NF-kappa B response in endothelial cells and inflammation in vivo.

BACKGROUND: Organs intended for transplantation are generally stored in the cold for better preservation of their function. However, following transplantation and reperfusion, the microvasculature of transplanted organs often proves to be activated. Extensive leukocyte adhesion and microthrombus formation contribute to failure of the transplanted organ. OBJECTIVES: In this study we analyzed cold-induced changes to the activation status of cultured endothelial cells, possibly contributing to organ failure. METHODS: We exposed human umbilical vein endothelial cells (HUVECs) to temperatures below 37 °C (mostly to 8 °C) for 30 min and upon rewarming to 37 °C kept incubating them for up to 24 h. We also in vivo locally exposed mice to cold. RESULTS: The exposure to low temperatures induced, in HUVECs, expression of the prothrombotic factors plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) and of the inflammatory adhesion molecules, E-selectin, intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Furthermore, upon rewarming for 30 min, we detected activation of the inflammatory NF-κB pathway, as measured by transient NF-κB translocation to the nucleus and IκBα degradation. Using butylated hydroxytoluene (BHT), a scavenger of reactive oxygen species (ROS), we further demonstrated that cold-induced NF-κB activation depends on ROS production. Local exposure to cold also, in vivo, induced ROS production and ICAM-1 expression and resulted in leukocyte infiltration. CONCLUSIONS: Our results point to a causative link between ROS production and NF-κB activation, suppression of which had been shown to be beneficial during hypothermic storage and subsequent rewarming of organs for transplantation.

The inflammatory mediator oncostatin M induces angiopoietin 2 expression in endothelial cells in vitro and in vivo.

OBJECTIVES: Members of the glycoprotein 130 (gp130) receptor-gp130 ligand family play a role in angiogenesis in different tissues. We tested the effect of this cytokine family on the angiopoietin (Ang)-Tie system, which is involved in blood vessel maturation, stabilization, and regression. RESULTS: Oncostatin M (OSM) increased Ang2 expression in human umbilical vein endothelial cells via Janus kinase/signal transducer and activator of transcription (JAK/STAT) and mitogen-activated protein (MAP) kinase activation. Furthermore, OSM induced Ang2 expression in macrovascular endothelial cells isolated from the human aorta and in microvascular endothelial cells isolated from human heart. Our in vivo experiments revealed that mRNA expression of Ang2 in hearts of mice injected with OSM increased significantly, and levels of OSM mRNA significantly correlated with mRNA levels of Ang2 in human hearts. In addition, OSM increased the expression of its own receptors, gp130 and OSM receptor, in endothelial cells in vitro and in mice in vivo, and levels of OSM mRNA significantly correlated with mRNA levels of gp130 and OSM receptor in human hearts. CONCLUSION: Our data, showing the effects of OSM on the Ang-Tie system in endothelial cells, in hearts of mice, and in human heart tissue, provide yet another link between inflammation and angiogenesis.

Vascular endothelial growth factor is induced by the inflammatory cytokines interleukin-6 and oncostatin m in human adipose tissue in vitro and in murine adipose tissue in vivo.

OBJECTIVES: It is believed that adipose tissue acts as an endocrine organ by producing inflammatory mediators and thereby contributes to the increased cardiovascular risk seen in obesity. A link between adipose tissue mass and angiogenesis has been suggested. Vascular endothelial growth factor (VEGF) seems to be implicated in this process. Members of the glycoprotein (gp)130 ligand family regulate VEGF expression in other cells. METHODS AND RESULTS: We used tissue explants as well as primary cultures of preadipocytes and adipocytes from human subcutaneous and visceral adipose tissue to investigate whether the gp130 ligands oncostatin M (OSM), interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and cardiotrophin-1 (CT-1) regulate VEGF expression in human adipose tissue. Human subcutaneous and visceral adipose tissue responded to treatment with IL-6 and OSM with a significant increase in VEGF production. Human preadipocytes were isolated from subcutaneous and visceral adipose tissue. Adipocyte-differentiation was induced by hormone-supplementation. All cell types responded to IL-6 and OSM with a robust increase in VEGF protein production and a similar increase in VEGF-specific mRNA. Furthermore, IL-1beta synergistically enhanced the effect of OSM on VEGF production. AG-490, a JAK/STAT inhibitor, abolished the OSM-dependent VEGF induction almost completely. In mice, IL-6 and OSM increased serum levels of VEGF and VEGF mRNA and vessel density in adipose tissue. CONCLUSION: We speculate that the inflammatory cytokines IL-6 and OSM might support angiogenesis during adipose tissue growth by upregulating VEGF.

Activation of nuclear factor-kappa B significantly contributes to lumen loss in a rabbit iliac artery balloon angioplasty model.

BACKGROUND: To investigate the contribution of inflammation to postangioplasty lumen loss, we used an adenoviral gene therapy approach to inhibit the central inflammatory mediator nuclear factor-kappaB (NF-kappaB) by overexpression of its natural inhibitor, IkappaBalpha. METHODS AND RESULTS: The adenovirus carrying human IkappaBalpha was applied immediately after balloon dilatation by a double-balloon catheter in a rabbit iliac artery restenosis model. Immunohistochemistry of IkappaBalpha revealed that mainly smooth muscle cells of the media but also cells of the adventitia were transduced and expressed the transgene IkappaB alpha for >/= 8 days. At this time point, intercellular adhesion molecule-1 (30%) and monocyte chemotactic protein-1 (50%) expression, as well as recruitment of macrophages into the wounded area (90%), were significantly reduced in IkappaB alpha-treated vessels. In addition, expression of inhibitor of apoptosis proteins was reduced and the percentage of apoptotic cells was increased compared with control-treated contralateral vessels. Animals killed 5 weeks after treatment exhibited a significantly reduced degree of lumen narrowing (P<0.02) on the side treated with adenovirus IkappaBalpha. The lumen gain of approximately 40% was due to positive remodeling. CONCLUSIONS: From these data, we conclude that balloon angioplasty-induced activation of NF-kappaB contributes to lumen loss likely via induction of an inflammatory response and a decrease in the rate of apoptosis. These data show for the first time that inflammation mediated by NF-kappaB is involved in postangioplasty lumen narrowing. Specific and more potent inhibitors of NF-kappaB might therefore be a useful therapeutic measure to improve clinical outcome after balloon dilatation.

Expression of the integrin alpha8beta1 during pulmonary and hepatic fibrosis.

The fibrotic response after diverse forms of injury is characterized by the accumulation of extracellular matrix proteins, proliferation of myofibroblast-like cells, and organ contraction. Myofibroblasts are key effector cells in the development of the fibrotic response. They contribute to fibrosis through both increased cell number (proliferation) and enhanced matrix synthesis. Integrins, a class of cell adhesion molecules, are mediators of cell-extracellular matrix protein interactions that are important in the proliferative and migratory response of cells to matrix proteins. We have previously cloned the human integrin subunit alpha8, documented its high expression in lung tissue, and established it as a receptor for the matrix proteins fibronectin, vitronectin, and tenascin. We now demonstrate that alveolar interstitial cells are the primary cell type expressing alpha8beta1 in the lung parenchyma. Expression of alpha8beta1 is concentrated primarily along the thinned extensions of cells and at the tips of filopodia. Because of its unique distribution in alveolar interstitial cells, we hypothesized that it may play a role in the fibrotic response after injury. In bleomycin-induced pulmonary fibrosis, there is increased expression of alpha8beta1 by interstitial fibroblasts, the majority of which coexpress alpha smooth muscle actin, a marker of tissue myofibroblasts. To establish a more general role for alpha8beta1 during organ fibrosis, we further examined its expression in two rat models of liver fibrosis. During hepatic injury due to either carbon tetrachloride injury or bile duct ligation, we demonstrate de novo expression of alpha8beta1 in activated hepatic stellate cells, the myofibroblast equivalent in liver. Taken together, the data localize alpha8beta1 to myofibroblast-like cells during wound healing and suggest that signal transduction through the alpha8beta1 integrin may contribute to the fibrotic response of organs to injury.

Hepatocyte growth factor increases expression of vascular endothelial growth factor and plasminogen activator inhibitor-1 in human keratinocytes and the vascular endothelial growth factor receptor flk-1 in human endothelial cells.

The pleiotropic growth factor hepatocyte growth factor/scatter factor (HGF/SF) has been implicated by clinical and experimental studies in repair mechanisms in different organs and tissues. However, no data on the impact of HGF/SF in wound healing in the skin are yet available. Proliferating and migrating keratinocytes play a major role in repair processes in the skin by closing the wound. Recent evidence gathered from studies that used gene-deficient mice has implicated the plasminogen activator (PA)/plasmin system in wound healing, which depends on controlled matrix degradation and deposition during cell migration and proliferation. Furthermore, keratinocytes are an important source of vascular endothelial growth factor (VEGF), which is a potent inducer of angiogenesis. In this study, we show that in human keratinocytes HGF/SF but not the related cytokine macrophage stimulating protein (MSP) significantly increases expression of VEGF and plasminogen activator inhibitor-1 (PAI-1) on the level of protein and mRNA. Furthermore, we demonstrate that HGF/SF increases the expression of the VEGF receptor flk-1 in human endothelial cells and that, in an angiogenesis co-culture assay of endothelial cells and keratinocytes, HGF/SF increases endothelial cell tube formation significantly. Therefore, we propose a role for HGF/SF in wound repair in the skin: HGF/SF--produced by activated fibroblasts--increases in keratinocytes the expression of PAI-1, which leads to increased matrix stability during the repair process and which could also limit activation of HGF/SF by proteases such as urokinase-type PA (u-PA) or tissue-type PA (t-PA). Furthermore HGF/SF also increases the expression of VEGF in these cells, thereby initiating angiogenesis in a paracrine manner. This effect would be enhanced by an increased responsiveness of endothelial cells toward VEGF, resulting from the HGF/SF-induced up-regulation of flk-1 on these cells.

Expression of the beta 6 integrin subunit in development, neoplasia and tissue repair suggests a role in epithelial remodeling.

The alpha v beta 6 integrin was identified in cultured epithelial cells and functions as a fibronectin receptor. We have now used monoclonal antibodies to determine in vivo expression patterns of the beta 6 subunit in normal and pathological human or primate tissues, and during experimental wound healing or induced lung injury. The results indicate that beta 6 expression is restricted to epithelia and is up-regulated in parallel with morphogenetic events, tumorigenesis, and epithelial repair. During development of the kidney, lung, and skin, we found that beta 6 is expressed by specific types of epithelial cells, whereas it is mostly undetectable in normal adult kidney, lung and skin. In contrast, we detected high-level expression in several types of carcinoma. For example, beta 6 is almost invariably neo-expressed in squamous cell carcinomas derived from the oral mucosa, often focally localized at the infiltrating edges of tumor islands. Expression of beta 6 is also upregulated in migrating keratinocytes at the wound edge during experimental epidermal wound healing. Similarly, beta 6 expression is induced in type II alveolar epithelial cells during lung injury caused by injection of live bacteria. We also observed beta 6 expression in adult lungs and kidneys at focal sites of subclinical inflammation, as well as in a variety of clinical specimens from patients with chronic or acute inflammation of the lungs or kidneys. From these findings and earlier results, we hypothesize that alpha v beta 6 affects cell spreading, migration and growth during reorganization of epithelia in development, tissue repair, and neoplasia.

Sequence and tissue distribution of the human integrin alpha 8 subunit: a beta 1-associated alpha subunit expressed in smooth muscle cells.

Integrins are a major family of cell adhesion molecules involved in cell-cell and cell-extracellular matrix interactions. Each integrin is a heterodimeric glycoprotein composed of an alpha and a beta subunit. We now report the cDNA sequence and distribution of a new human integrin alpha subunit. This sequence is 78% identical to the previously reported chicken alpha 8 integrin sequence. Thus, we have designated this subunit as human alpha 8. By northern blot analysis, an alpha 8 probe detects two mRNA species of approximately 6.5 and 4.0 kb in neuroglioma H4 cells. An anti-alpha 8 polyclonal antibody precipitates a protein complex containing the beta 1 subunit associated with the putative alpha 8 subunit, which has an apparent molecular mass of 180 kDa (non-reduced) or 155 kDa and 25 kDa (reduced). Immunohistochemistry with anti-alpha 8 polyclonal antibody in adult rat tissues shows prominent staining in vascular and visceral smooth muscle. In addition, the antibody strongly stained kidney mesangial cells and a cell type in the alveolar wall of the lungs, most likely corresponding to alveolar myofibroblasts. These results suggest that in adult mammalian tissues, alpha 8 is predominantly expressed in smooth muscle and smooth muscle-like contractile cells.

Restricted distribution of integrin beta 6 mRNA in primate epithelial tissues.

Integrins are cell adhesion receptors that mediate cell-extracellular matrix and cell-cell interactions. Each integrin consists of two glycoprotein subunits (alpha and beta). We have previously described a novel integrin beta-subunit, beta 6, which is expressed in cultured epithelial cells. beta 6 can associate with alpha v to form the fibronectin-binding heterodimer alpha v beta 6. Here we report the tissue distribution of beta 6 integrin mRNA determined by in situ hybridization of a beta 6 cRNA probe with representative frozen tissue sections from a rhesus monkey tissue bank. We detected beta 6 mRNA exclusively in epithelial cells. However, beta 6 mRNA expression varied greatly among different epithelia. High levels of beta 6 mRNA were found only in two very specialized epithelial cell types: a portion of the kidney tubule epithelium, termed macula densa, and the endometrial epithelium of secretory phase uterus. In the endometrium, beta 6 expression was highest in the differentiated epithelium of functional layer glands, suggesting that beta 6 expression can be regulated in a differentiation-dependent manner. beta 6 expression may also depend on the stage in the estrous cycle, since we found much lower beta 6 mRNA levels in a specimen of proliferative phase endometrium. Epithelium in several other tissues, including salivary gland ducts, gall bladder, and epididymis, contained detectable levels of beta 6 mRNA, albeit much lower than in macula densa and endometrium. In other epithelia, including skin and lung, beta 6 mRNA was undetectable. Taken together, these results suggest that in normal adult primates beta 6 expression is regulated in a cell type-specific manner, restricted to a few epithelial tissues.