Reactive oxygen and NF-kappaB in VEGF-induced migration of human vascular smooth muscle cells.

Migration and proliferation of vascular smooth muscle cells (VSMC) contribute to angiogenesis and the lesions of atherosclerosis. Since, vascular endothelial growth factor (VEGF) is overexpressed by VSMC in intima of atherosclerotic human coronary arteries, we determined if VEGF could stimulate VSMC migration and the intracellular signals involved. VEGF induced VSMC migration but had no significant activity on proliferation. VEGF increased intracellular reactive oxygen species (ROS), NF-kappaB activation and IL-6 expression. Blockade of the generation of intracellular ROS by antioxidants inhibited VEGF-induced NF-kappaB activation, IL-6 expression, and cell migration indicating that generation of ROS was required for NF-kappaB activation and the chemotactic activity of VEGF. Expression of a mutated, nondegradable form of inhibitor of NF-kappaB (IkappaB-alphaM) suppressed VEGF-triggered activation of NF-kappaB and upregulation of IL-6 as well as VSMC migration. Neutralization of IL-6 by its antibody significantly attenuated the migration stimulated by VEGF. Collectively, our data provide the first evidence that intracellular ROS and NF-kappaB are required for VEGF-mediated smooth muscle cell migration. Further, IL-6 induced by VEGF is involved in the ability of the growth factor to stimulate migration.     

Rapamycin antagonizes NF-kappaB nuclear translocation activated by TNF-alpha in primary vascular smooth muscle cells and enhances apoptosis

Several lines of evidence support the view that rapamycin inhibits NF-kappaB. TNF-alpha, a potent inducer of NF-kappaB, is released after artery injury (e.g., balloon angioplasty) and plays an important role in inflammation and restenosis. We investigated the effect of rapamycin on NF-kappaB activation and apoptosis in vascular smooth muscle cells (VSMCs) stimulated with TNF-alpha. Using EMSA, we found that TNF-alpha caused NF-kappaB nuclear translocation in VSMCs after 1 h of incubation. Rapamycin inhibited IkappaBalpha degradation, thereby preventing nuclear translocation. Activation of NF-kappaB was accompanied by an increase of Bcl-xL and Bfl-1/A1 proteins, detected by Western blot assay, whereas rapamycin prevented the TNF-alpha-induced enhancement of these antiapoptotic proteins. The extent of apoptosis of VSMCs exposed to TNF-alpha was significantly enhanced by rapamycin. The effect of rapamycin appeared to be independent of the phosphatidylinositol 3-kinase/Akt-protein kinase B survival pathway, because the phosphatidylinositol 3-kinase inhibitor wortmannin neither prevented IkappaBalpha degradation nor increased apoptosis of cells incubated with TNF-alpha. Finally, we demonstrate that the large immunophilin FK-506 binding protein FKBP51 is essential for TNF-alpha-induced NF-kappaB activation in VSMCs. Our findings show that rapamycin inhibits NF-kappaB activation and acts in concert with TNF-alpha in induction of VSMC apoptosis.     

Phosphatidylinositol 3-kinase/Akt signaling mediates interleukin-32alpha induction in human pancreatic periacinar myofibroblasts

Interleukin (IL)-32 is a recently described proinflammatory cytokine, characterized by the induction of nuclear factor (NF)-kappaB activation. We studied IL-32alpha expression in human pancreatic periacinar myofibroblasts, which play important roles in the regulation of extracellular matrix metabolism and inflammatory responses in the pancreas. IL-32alpha protein expression was evaluated by Western blot analyses, and IL-32alpha mRNA expression was analyzed by Northern blot and real-time PCR analyses. IL-32alpha mRNA was weakly expressed without a stimulus, and its expression was markedly enhanced by IL-1beta, IFN-gamma, and TNF-alpha. IL-1beta, IFN-gamma, and TNF-alpha enhanced intracellular accumulation of IL-32alpha protein, but IL-32alpha was not detected in supernatants. Each cytokine dose and time dependently induced IL-32alpha mRNA expression. An inhibitor of phosphatidylinositol 3-kinase (LY294002) significantly suppressed IL-1beta-, IFN-gamma-, and TNF-alpha-induced IL-32alpha mRNA expression, although MAPK inhibitors had no effect. Akt activation in response to these cytokines was confirmed by Western blot. Furthermore, LY294002 suppressed both IL-1beta- and TNF-alpha-induced NF-kappaB activation and IL-1beta-, TNF-alpha-, and IFN-gamma-induced activated protein-1 (AP-1) activation. Blockade of NF-kappaB and AP-1 activation by an adenovirus expressing a stable mutant form of IkappaBalpha and a dominant negative mutant of c-Jun markedly suppressed IL-1beta-, IFN-gamma-, and/or TNF-alpha-induced IL-32alpha mRNA expression. Human pancreatic periacinar myofibroblasts expressed IL-32alpha in response to IL-1beta, TNF-alpha, and IFN-gamma. IL-32alpha mRNA expression is dependent on interactions between the phosphatidylinositol 3-kinase/Akt-pathway and the NF-kappaB/AP-1 system.     

Resveratrol attenuates TNF-alpha-induced activation of coronary arterial endothelial cells: role of NF-kappaB inhibition

Epidemiological studies suggest that Mediterranean diets rich in resveratrol are associated with reduced risk of coronary artery disease. However, the mechanisms by which resveratrol exerts its cardioprotective effects are not completely understood. Because TNF-alpha-induced endothelial activation and vascular inflammation play a critical role in vascular aging and atherogenesis, we evaluated whether resveratrol inhibits TNF-alpha-induced signal transduction in human coronary arterial endothelial cells (HCAECs). We found that TNF-alpha significantly increased adhesiveness of the monocytic THP-1 cells to HCAECs, an effect that could be inhibited by pretreatment with resveratrol and the NF-kappaB inhibitor pyrrolidine dithiocarbamate. Previously, we found that TNF-alpha activates NAD(P)H oxidases, and our recent data showed that TNF-alpha-induced endothelial activation was prevented by the NAD(P)H oxidase inhibitor apocynin or catalase plus SOD. Resveratrol also inhibited H(2)O(2)-induced monocyte adhesiveness. Using a reporter gene assay, we found that, in HCAECs, TNF-alpha significantly increased NF-kappaB activity, which could be inhibited by resveratrol (>50% inhibition at 10(-6) mol/l) and pyrrolidine dithiocarbamate. Resveratrol also inhibited TNF-alpha-induced, NF-kappaB-driven luciferase expression in rat aortas electroporated with the reporter gene construct. In TNF-alpha-treated HCAECs, resveratrol (in the submicromolar range) significantly attenuated expression of NF-kappaB-dependent inflammatory markers inducible nitric oxide synthase, IL-6, bone morphogenetic protein-2, ICAM-1, and VCAM. Thus resveratrol at nutritionally relevant concentrations inhibits TNF-alpha-induced NF-kappaB activation and inflammatory gene expression and attenuates monocyte adhesiveness to HCAECs. We propose that these anti-inflammatory actions of resveratrol are responsible, at least in part, for its cardioprotective effects.     

Fas ligand induces cell-autonomous NF-kappaB activation and interleukin-8 production by a mechanism distinct from that of tumor necrosis factor-alpha

Fas ligand (FasL) has been well characterized as a death factor. However, recent studies revealed that FasL possesses inflammatory activity. Here we found that FasL induces production of the inflammatory chemokine IL-8 without inducing apoptosis in HEK293 cells. Reporter gene assays involving wild-type and mutated IL-8 promoters and NF-kappaB- and AP-1 reporter constructs indicated that an FasL-induced NF-kappaB and AP-1 activity are required for maximal promoter activity. FasL induced NF-kappaB activation with slower kinetics than did TNF-alpha, yet this response was cell autonomous and not mediated by secondary paracrine factors. The death domain of Fas, FADD, and caspase-8 were required for NF-kappaB activation by FasL. A dominant-negative mutant of IKKgamma inhibited the FasL-induced NF-kappaB activation. However, TRADD and RIP, which are essential for the TNF-alpha-induced NF-kappaB activation, were not involved in the FasL-induced NF-kappaB activation. Moreover, CLARP/FLIP inhibited the FasL- but not the TNF-alpha-induced NF-kappaB activation. These results show that FasL induces NF-kappaB activation and IL-8 production by a novel mechanism, distinct from that of TNF-alpha. In addition, we found that mouse FADD had a dominant-negative effect on the FasL-induced NF-kappaB activation in HEK293 cells, which may indicate a species difference between human and mouse in the FasL-induced NF-kappaB activation.     

Involvement of interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis.

Tumor necrosis factor alpha (TNF-alpha) is a macrophage/monocyte-derived polypeptide which modulates the expression of various genes in vascular endothelial cells and induces angiogenesis. However, the underlying mechanism by which TNF-alpha mediates angiogenesis is not completely understood. In this study, we assessed whether TNF-alpha-induced angiogenesis is mediated through TNF-alpha itself or indirectly through other TNF-alpha-induced angiogenesis-promoting factors. Cellular mRNA levels of interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and their receptors were increased after the treatment of human microvascular endothelial cells with TNF-alpha (100 U/ml). TNF-alpha-dependent tubular morphogenesis in vascular endothelial cells was inhibited by the administration of anti-IL-8, anti-VEGF, and anti-bFGF antibodies, and coadministration of all three antibodies almost completely abrogated tubular formation. Moreover, treatment with Sp1, NF-kappaB, and c-Jun antisense oligonucleotides inhibited TNF-alpha-dependent tubular morphogenesis by microvascular endothelial cells. Administration of a NF-kappaB antisense oligonucleotide almost completely inhibited TNF-alpha-dependent IL-8 production and partially abrogated TNF-alpha-dependent VEGF production, and an Sp1 antisense sequence partially inhibited TNF-alpha-dependent production of VEGF. A c-Jun antisense oligonucleotide significantly inhibited TNF-alpha-dependent bFGF production but did not affect the production of IL-8 and VEGF. Administration of an anti-IL-8 or anti-VEGF antibody also blocked TNF-alpha-induced neovascularization in the rabbit cornea in vivo. Thus, angiogenesis by TNF-alpha appears to be modulated through various angiogenic factors, both in vitro and in vivo, and this pathway is controlled through paracrine and/or autocrine mechanisms.     

Effects of antioxidant and nitric oxide on chemokine production in TNF-alpha-stimulated human dermal microvascular endothelial cells

Chemokines have been implicated convincingly in the driving of leukocyte emigration in different inflammatory reactions. Multiple signaling mechanisms are reported to be involved in intracellular activation of chemokine expression in vascular endothelial cells by various stimuli. Nevertheless, redox-regulated mechanisms of chemokine expression in human dermal microvascular endothelial cells (HDMEC) remain unclear. This study examined the effects of pyrrolidine dithiocarbamate (PDTC, 0.1 mM) and spermine NONOate (Sper-NO, 1 mM) on the secretion and gene expression of chemokines, interleukin (IL)-8, monocyte chemotactic protein (MCP)-1, regulated upon activation normal T cell expressed and secreted (RANTES), and eotaxin. This study also addresses PDTC and Sper-NO effects on activation of nuclear factor kappa B (NF-kappaB) induced by TNF-alpha (10 ng/ml). Treatment with TNF-alpha for 8 h significantly increased secretion of IL-8, MCP-1, and RANTES, but not of eotaxin, in cultured HDMEC. Up-regulation of these chemokines was suppressed significantly by pretreatment with PDTC or Sper-NO for 1 h, but not by 1 mM 8-bromo-cyclic GMP. The mRNA accumulation of IL-8, MCP-1, RANTES, and eotaxin, and activation of NF-kappaB were induced by TNF-alpha for 2 h; all were suppressed significantly by the above two pretreatments. These findings indicate that both secretion and mRNA accumulation of IL-8, MCP-1, and RANTES in HDMEC induced by TNF-alpha are inhibited significantly by pretreatment with PDTC or Sper-NO, possibly via blocking redox-regulated NF-kappaB activation. These results suggest that restoration of the redox balance using antioxidant agents or nitric oxide pathway modulators may offer new opportunities for therapeutic interventions in inflammatory skin diseases.