Oxidative stress augments the production of matrix metalloproteinase-1, cyclooxygenase-2, and prostaglandin E2 through enhancement of NF-kappa B activity in lipopolysaccharide-activated human primary monocytes

The excessive production of reactive oxidative species (ROS) associated with inflammation leads to a condition of oxidative stress. Cyclooxygenase-2 (COX-2), PGE(2), and matrix metalloproteinases (MMPs) are important mediators during the process of inflammation. In this paper we report on studies examining how the ROS hydrogen peroxide (H(2)O(2)) affects the production of MMP-1, COX-2, and PGE(2). Addition of H(2)O(2) to LPS-activated monocytes, but not naive monocytes, caused a significant enhancement of the LPS-induced production of MMP-1, COX-2, and PGE(2). The mechanism by which H(2)O(2) increased these mediators was through enhancement of IkappaBalpha degradation, with subsequent increases in NF-kappaB activation and NF-kappaB p50 translocation to the nucleus. The effects of H(2)O(2) on IkappaBalpha degradation, NF-kappaB activation, and NF-kappaB p50 localization to the nucleus were demonstrated through studies of coimmunoprecipitation of IkappaBalpha with p50, ELISA of NF-kappaB p65 activity, and Western blot analysis of the nuclear fraction extract for p50. The key role for NF-kappaB in this process was demonstrated by the ability of MG-132 or lactacystin (proteasome inhibitors) to block the enhanced production of MMP-1, COX-2, and PGE(2). In contrast, indomethacin, which inhibited PGE(2) production, partially blocked the enhanced MMP-1 production. Moreover, although PGE(2) restored MMP-1 production in indomethacin-treated monocyte cultures; it failed to significantly restore MMP-1 production in proteasome inhibitor-treated cultures. Thus, in the presence of LPS and H(2)O(2), NF-kappaB plays a dominate role in the regulation of MMP-1, COX-2, and PGE(2) expression.     

Novel role of p66Shc in ROS-dependent VEGF signaling and angiogenesis in endothelial cells

p66Shc, a longevity adaptor protein, is demonstrated as a key regulator of reactive oxygen species (ROS) metabolism involved in aging and cardiovascular diseases. Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration and proliferation primarily through the VEGF receptor-2 (VEGFR2). We have shown that ROS derived from Rac1-dependent NADPH oxidase are involved in VEGFR2 autophosphorylation and angiogenic-related responses in ECs. However, a role of p66Shc in VEGF signaling and physiological responses in ECs is unknown. Here we show that VEGF promotes p66Shc phosphorylation at Ser36 through the JNK/ERK or PKC pathway as well as Rac1 binding to a nonphosphorylated form of p66Shc in ECs. Depletion of endogenous p66Shc with short interfering RNA inhibits VEGF-induced Rac1 activity and ROS production. Fractionation of caveolin-enriched lipid raft demonstrates that p66Shc plays a critical role in VEGFR2 phosphorylation in caveolae/lipid rafts as well as downstream p38MAP kinase activation. This in turn stimulates VEGF-induced EC migration, proliferation, and capillary-like tube formation. These studies uncover a novel role of p66Shc as a positive regulator for ROS-dependent VEGFR2 signaling linked to angiogenesis in ECs and suggest p66Shc as a potential therapeutic target for various angiogenesis-dependent diseases.     

Differential roles of vascular endothelial growth factor receptors 1 and 2 in dendritic cell differentiation

Impaired Ag-presenting function in dendritic cells (DCs) due to abnormal differentiation is an important mechanism of tumor escape from immune control. A major role for vascular endothelial growth factor (VEGF) and its receptors, VEGFR1/Flt-1 and VEGFR2/KDR/Flk-1, has been documented in hemopoietic development. To study the roles of each of these receptors in DC differentiation, we used an in vitro system of myeloid DC differentiation from murine embryonic stem cells. Exposure of wild-type, VEGFR1(-/-), or VEGFR2(-/-) embryonic stem cells to exogenous VEGF or the VEGFR1-specific ligand, placental growth factor, revealed distinct roles of VEGF receptors. VEGFR1 is the primary mediator of the VEGF inhibition of DC maturation, whereas VEGFR2 tyrosine kinase signaling is essential for early hemopoietic differentiation, but only marginally affects final DC maturation. SU5416, a VEGF receptor tyrosine kinase inhibitor, only partially rescued the mature DC phenotype in the presence of VEGF, suggesting the involvement of both tyrosine kinase-dependent and independent inhibitory mechanisms. VEGFR1 signaling was sufficient for blocking NF-kappaB activation in bone marrow hemopoietic progenitor cells. VEGF and placental growth factor affect the early stages of myeloid/DC differentiation. The data suggest that therapeutic strategies attempting to reverse the immunosuppressive effects of VEGF in cancer patients might be more effective if they specifically targeted VEGFR1.     

Cytokine-induced activation of nuclear factor-kappa B is inhibited by hydrogen peroxide through oxidative inactivation of IkappaB kinase

Rapid activation of the IkappaB kinase (IKK) complex is considered an obligatory step in the activation of nuclear factor-kappaB (NF-kappaB) in response to diverse stimuli. Since oxidants have been implicated in the regulation of NF-kappaB, the focus of the present study was the activation of IKK by tumor necrosis factor alpha (TNFalpha) in the presence or absence of hydrogen peroxide (H(2)O(2)). Exposure of mouse alveolar epithelial cells to H(2)O(2) was not sufficient to activate IKK, degrade IkappaBalpha, or activate NF-kappaB. In contrast, TNFalpha induced IKK activity rapidly and transiently resulting in IkappaBalpha degradation and NF-kappaB activation. Importantly, in the presence of H(2)O(2), the ability of TNFalpha to induce IKK activity was markedly decreased and resulted in prevention of IkappaBalpha degradation and NF-kappaB activation. Neither tyrosine kinases nor phosphatidylinositol 3-kinases, known regulators of NF-kappaB by oxidants, were involved in IKK inhibition by H(2)O(2). Direct addition of H(2)O(2) to the immunoprecipitated IKK complex inhibited enzyme activity. Inhibition of IKK activity by H(2)O(2) was associated with direct oxidation of cysteine residues present in the IKK complex and occurred only in enzymatically active IKK. In contrast to previously published observations, our findings demonstrate that the oxidant H(2)O(2) reduces NF-kappaB activation by inhibiting activated IKK activity.     

The Bartonella henselae VirB/Bep system interferes with vascular endothelial growth factor (VEGF) signalling in human vascular endothelial cells

The vasculotropic pathogen Bartonella henselae (Bh) intimately interacts with human endothelial cells (ECs) and subverts multiple cellular functions. Here we report that Bh specifically interferes with vascular endothelial growth factor (VEGF) signalling in ECs. Bh infection abrogated VEGF-induced proliferation and wound closure of EC monolayers as well as the capillary-like sprouting of EC spheroids. On the molecular level, Bh infection did not alter VEGF receptor 2 (VEGFR2) expression or cell surface localization, but impeded VEGF-stimulated phosphorylation of VEGFR2 at tyrosine(1175) . Consistently, we observed that Bh infection diminished downstream events of the tyrosine(1175) -dependent VEGFR2-signalling pathway leading to EC proliferation, i.e. phospholipase-Cγ activation, cytosolic calcium fluxes and mitogen-activated protein kinase ERK1/2 phosphorylation. Pervanadate treatment neutralized the inhibitory activity of Bh on VEGF signalling, suggesting that Bh infection may activate a phosphatase that alleviates VEGFR2 phosphorylation. Inhibition of VEGFR2 signalling by Bh infection was strictly dependent on a functional VirB type IV secretion system and thereby translocated Bep effector proteins. The data presented in this study underscore the role of the VirB/Bep system as important factor controlling EC proliferation in response to Bh infection; not only as previously reported by counter-acting an intrinsic bacterial mitogenic stimulus, but also by restricting the exogenous angiogenic stimulation by Bh-induced VEGF.     

The effect of age on cyclooxygenase-2 gene expression: NF-kappaB activation and IkappaBalpha degradation

Increased oxidative stress resulting in the activation of NF-kappaB is thought to play a crucial role in the expression of the cyclooxygenase-2 (COX-2), which is the key enzyme in proinflammatory prostanoid synthesis. In the current study, we investigated whether the aging process affects the status of the redox-sensitive NF-kappaB in rat kidney, and how this age-related modulation is related to COX-2 gene expression and COX-derived reactive oxygen species (ROS). We found that the aging process strongly enhanced the activation of NF-kappaB and its DNA-binding activity with an increased ROS status. Accompanied with the change in the NF-kappaB activity was a decreased IkappaBalpha as confirmed by the increased nuclear p65 protein. Thus, these data strongly indicated that the aging process increases NF-kappaB activity by downregulating IkappaBalpha. A closer examination further revealed that age-related oxidative status correlated with the increased COX-derived prostanoid biosynthetic process is mediated by the increased NF-kappaB-regulated COX activity. This increase in NF-kappaB activity was accompanied by the increased COX-2 mRNA and protein levels. Based on these data, we concluded that the age-related increase in redox-sensitive NF-kappaB translocation and binding activities are associated with increased ROS, and further that this transactivation was modulated by the age-related decrease of IkappaBalpha.     

Thalidomide suppresses NF-kappa B activation induced by TNF and H2O2, but not that activated by ceramide, lipopolysaccharides, or phorbol ester

Thalidomide ([+]-alpha-phthalimidoglutarimide), a psychoactive drug that readily crosses the blood-brain barrier, has been shown to exhibit anti-inflammatory, antiangiogenic, and immunosuppressive properties through a mechanism that is not fully established. Due to the central role of NF-kappaB in these responses, we postulated that thalidomide mediates its effects through suppression of NF-kappaB activation. We investigated the effects of thalidomide on NF-kappaB activation induced by various inflammatory agents in Jurkat cells. The treatment of these cells with thalidomide suppressed TNF-induced NF-kappaB activation, with optimum effect occurring at 50 microg/ml thalidomide. These effects were not restricted to T cells, as other hematopoietic and epithelial cell types were also inhibited. Thalidomide suppressed H(2)O(2)-induced NF-kappaB activation but had no effect on NF-kappaB activation induced by PMA, LPS, okadaic acid, or ceramide, suggesting selectivity in suppression of NF-kappaB. The suppression of TNF-induced NF-kappaB activation by thalidomide correlated with partial inhibition of TNF-induced degradation of an inhibitory subunit of NF-kappaB (IkappaBalpha), abrogation of IkappaBalpha kinase activation, and inhibition of NF-kappaB-dependent reporter gene expression. Thalidomide abolished the NF-kappaB-dependent reporter gene expression activated by overexpression of TNFR1, TNFR-associated factor-2, and NF-kappaB-inducing kinase, but not that activated by the p65 subunit of NF-kappaB. Overall, our results clearly demonstrate that thalidomide suppresses NF-kappaB activation specifically induced by TNF and H(2)O(2) and that this may contribute to its role in suppression of proliferation, inflammation, angiogenesis, and the immune system.     

Tumor necrosis factor and reactive oxygen species cooperative cytotoxicity is mediated via inhibition of NF-kappaB

BACKGROUND: Tumor necrosis factor alpha (TNFalpha) plays a key role in pathogenesis of brain injury. However, TNFalpha exhibits no cytotoxicity in primary cultures of brain cells. This discrepancy suggests that other pathogenic stimuli that exist in the setting of brain injury precipitate TNFalpha cytotoxicity. The hypothesis was tested that reactive oxygen species (ROS), that are released early after brain injury, act synergistically with TNFalpha in causing cell death. MATERIALS AND METHODS: Cultured human and rat brain capillary endothelial cells (RBEC), and cortical astrocytes were treated with TNFalpha alone or together with different doses of H2O2, and apoptotic cell death and DNA fragmentation were measured by means of 3'-OH-terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and Hoechst fluorescence assay, respectively. The effect of H2O2 on TNFalpha-induced activation of nuclear factor kappa B (NF-kappaB) was measured by Western blots of cytoplasmic and nuclear extracts of RBEC using anti-inhibitor of NF-kappaB (IkappaB) and anti-p65 subunit of NF-kappaB antibodies. Nuclear translocation of NF-kappaB was investigated by immunofluorescent staining of RBEC with anti-p65 antibodies. RESULTS: TNFalpha alone had no cytotoxic effect in brain endothelial cells and astrocytes at concentrations up to 100 ng/ml. Co-treatment with 5-10 microM of H2O2 caused a two-fold increase in the number of apoptotic cells 24 hr later. Similar doses (1-3 microM) of H2O2 initiated early DNA fragmentation. H2O2 inhibited TNFalpha-induced accumulation of p65 in the nucleus, although it had no effect on degradation of the IkappaB in cytoplasm. Immunostaining confirmed that H2O2 inhibited p65 transport to the nucleus. CONCLUSIONS: Reactive oxygen species could act synergistically with TNFalpha in causing cytotoxicity via inhibition of a cytoprotective branch of TNFalpha signaling pathways, which starts with NF-kappaB activation.     

Aspirin prevents apoptosis and NF-kappaB activation induced by H2O2 in hela cells

The classical pathway of nuclear factor-kappa B (NF-kappaB) activation by several inducers mainly involves the phosphorylation of IkappaBalpha by a signalsome complex composed of IkappaBalpha kinases (IKKalpha and IKKbeta). However, in some cell types hydrogen peroxide (H2O2) has been shown to activate an alternative pathway that does not involve the classical signalsome activation process. In this study, we demonstrate that H2O2 induced NF-kappaB activation in HeLa cells through phosphorylation and degradation of IkappaB proteins as shown by immunblot analysis. Our studies reveal that a commonly used non-steroid anti-inflammatory drug, acetylsalicylic acid (aspirin) prevents H2O2-induced NF-kappaB activation in a dose-dependent manner through inhibition of phosphorylation and degradation of IkappaBalpha and IkappaBbeta. Differential staining and DNA fragmentation analysis also show that aspirin preloading of HeLa cells also prevents H2O2-induced apoptosis in a dose-dependent manner with maximum efficiency at 10 mM concentration. Additionally, aspirin effectively prevents caspase-3 and caspase-9 (cysteinyl aspartate-specific proteases) activation by H2O2. These results suggest that NF-kappaB activation is involved in H2O2-induced apoptosis and aspirin may inhibit both processes simultaneously.     

Suppressive effect of reactive oxygen species on CD40-induced B cell activation

Reactive oxygen species (ROS) produced by the innate immune system work as effectors to destroy pathogens and to control cellular responses. However, their role in the adaptive immune response remains unclear. Here we studied the effect of exogenous ROS on CD40-induced B cell activation. H2O2 treatment inhibited CD40-induced immunoglobulin production of B cells, DNA binding of NF-kappaB, IkappaBalpha degradation and IKK phosphorylation. On the other hand, H2O2 treatment did not induce obvious B cell death after 30 min of stimulation. Although the ligation of anti-CD40 antibody was not disturbed by H2O2, TRAF2 recruitment to CD40 was inhibited. These results suggest that exogenous ROS play a negative role in CD40 signaling during B cell activation.     

A quantitative study of NF-kappaB activation by H2O2: relevance in inflammation and synergy with TNF-alpha

Although the germicide role of H(2)O(2) released during inflammation is well established, a hypothetical regulatory function, either promoting or inhibiting inflammation, is still controversial. In particular, after 15 years of highly contradictory results it remains uncertain whether H(2)O(2) by itself activates NF-kappaB or if it stimulates or inhibits the activation of NF-kappaB by proinflammatory mediators. We investigated the role of H(2)O(2) in NF-kappaB activation using, for the first time, a calibrated and controlled method of H(2)O(2) delivery--the steady-state titration--in which cells are exposed to constant, low, and known concentrations of H(2)O(2). This technique contrasts with previously applied techniques, which disrupt cellular redox homeostasis and/or introduce uncertainties in the actual H(2)O(2) concentration to which cells are exposed. In both MCF-7 and HeLa cells, H(2)O(2) at extracellular concentrations up to 25 microM did not induce significantly per se NF-kappaB translocation to the nucleus, but it stimulated the translocation induced by TNF-alpha. For higher H(2)O(2) doses this stimulatory role shifts to an inhibition, which may explain published contradictory results. The stimulatory role was confirmed by the observation that 12.5 microM H(2)O(2), a concentration found during inflammation, increased the expression of several proinflammatory NF-kappaB-dependent genes induced by TNF-alpha (e.g., IL-8, MCP-1, TLR2, and TNF-alpha). The same low H(2)O(2) concentration also induced the anti-inflammatory gene coding for heme oxygenase-1 (HO-1) and IL-6. We propose that H(2)O(2) has a fine-tuning regulatory role, comprising both a proinflammatory control loop that increases pathogen removal and an anti-inflammatory control loop, which avoids an exacerbated harmful inflammatory response.     

BMPs promote proliferation and migration of endothelial cells via stimulation of VEGF-A/VEGFR2 and angiopoietin-1/Tie2 signalling

The differentiation, growth, and survival of endothelial cells (ECs) are regulated by multiple signalling pathways, such as vascular endothelial growth factors (VEGFs) and angiopoietins through their receptor tyrosine kinases, VEGF receptor (VEGFR) 2 and Tie2, respectively. Bone morphogenetic proteins (BMPs), members of the transforming growth factor (TGF)-beta family, have been implicated in the development and maintenance of vascular systems. However, their effects on EC proliferation remain to be elucidated. In the present study, we show that BMPs induce the proliferation and migration of mouse embryonic stem cell (ESC)-derived endothelial cells (MESECs) and human microvascular endothelial cells (HMECs). Addition of BMP-4 to culture induced significant proliferation and migration of both types of ECs. BMP-4 also increased the expression and phosphorylation of VEGFR2 and Tie2. These findings suggest that BMP signalling activates endothelium via activation of VEGF/VEGFR2 and Angiopoietin/Tie2 signalling.     

Reactive oxygen species as mediators of angiogenesis signaling. Role of NAD(P)H oxidase

Angiogenesis, a process of new blood vessel growth, contributes to various pathophysiologies such as cancer, diabetic retinopathy and atherosclerosis. Accumulating evidence suggests that cardiovascular diseases are associated with increased oxidative stress in blood vessels. Reactive oxygen species (ROS) such as superoxide and H2O2 cause blood vessels to thicken, produce inflammation in the vessel wall, and thus are regarded as "risk factors" for vascular disease, whereas ROS also act as signaling molecules in many aspects of growth factor-mediated physiological responses. Recent reports suggest that ROS play an important role in angiogenesis; however, its underlying molecular mechanisms remain unknown. Vascular endothelial growth factor (VEGF) induces angiogenesis by stimulating endothelial cell (EC) proliferation and migration primarily through the receptor tyrosine kinase VEGF receptor2 (Flk1/KDR). VEGF binding initiates tyrosine phosphorylation of KDR, which results in activation of downstream signaling enzymes including ERK1/2, Akt and eNOS, which contribute to angiogenic-related responses in EC. Importantly, the major source of ROS in EC is a NAD(P)H oxidase and EC express all the components of phagocytic NAD(P)H oxidase including gp91phox, p22phox, p47phox, p67phox and the small G protein Rac1. We have recently demonstrated that ROS derived from NAD(P)H oxidase are critically important for VEGF signaling in vitro and angiogenesis in vivo. Furthermore, a peptide hormone, angiotensin II, a major stimulus for vascular NAD(P)H oxidase, also plays an important role in angiogenesis. Because EC migration and proliferation are primary features of the process of myocardial angiogenesis, we would like to focus on the recent progress that has been made in the emerging area of NAD(P)H oxidase-derived ROS-dependent signaling in ECs, and discuss the possible roles in angiogenesis. Understanding these mechanisms may provide insight into the components of NAD(P)H oxidase as potential therapeutic targets for treatment of angiogenesis-dependent diseases such as cancer and atherosclerosis and for promoting myocardial angiogenesis in ischemic heart diseases.     

Phosphorylation of Dab2 is involved in inhibited VEGF‐VEGFR‐2 signaling induced by downregulation of syndecan‐1 in glomerular endothelial cell

Disabled‐2 (Dab2) and PAR‐3 (partitioning defective 3) are reported to play critical roles in maintaining retinal microvascular endothelial cells biology by regulating VEGF‐VEGFR‐2 signaling. The role of Dab2 and PAR‐3 in glomerular endothelial cell (GEnC) is unclear. In this study, we found that, no matter whether with vascular endothelial growth factor (VEGF) treatment or not, decreased expression of Dab2 could lead to cell apoptosis by preventing activation of VEGF‐VEGFR‐2 signaling in GEnC, accompanied by reduced membrane VEGFR‐2 expression. And silencing of PAR‐3 gene expression caused increased apoptosis of GEnC by inhibiting activation of VEGF‐VEGFR‐2 signaling and membrane VEGFR‐2 expression. In our previous research, we found that the silencing of syndecan‐1 gene expression inhibited VEGF‐VEGFR‐2 signaling by modulating internalization of VEGFR‐2. And our further research demonstrated that downregulation of syndecan‐1 lead to no significant change in the expression of Dab2 and PAR‐3 both at messenger RNA and protein levels in GEnC, while phosphorylation of Dab2 was significantly increased in GEnC transfected with Dab2 small interfering RNA (siRNA) compared with control siRNA. Atypical protein kinase C (aPKC) could induce phosphorylation of Dab2, thus negatively regulating VEGF‐VEGFR‐2 signaling. And we found that decreased expression of syndecan‐1 lead to activation of aPKC, and aPKC inhibitor treatment could block phosphorylation of Dab2 in GEnC. Besides, aPKC inhibitor treatment could activate VEGF‐VGEFR‐2 signaling in GEnC transfected with syndecan‐1 siRNA in a dose‐dependent manner. In conclusion, we speculated that phosphorylation of Dab2 is involved in preventing activation of VEGF‐VEGFR‐2 signaling in GEnC transfected with syndecan‐1 siRNA. This provides a new target for the therapy of GEnC injury and kidney disease.     

NADPH oxidase modulates myocardial Akt, ERK1/2 activation, and angiogenesis after hypoxia-reoxygenation

Recent studies have demonstrated that reactive oxygen species (ROS) mediate myocardial ischemia-reperfusion (I/R) and angiogenesis via the mitogen-activated protein kinases and the serine-threonine kinase Akt/protein kinase B pathways. NADPH oxidases are major sources of ROS in endothelial cells and cardiomyocytes. In the present study, we investigated the role of NADPH oxidase-derived ROS in hypoxia-reoxygenation (H/R)-induced Akt and ERK1/2 activation and angiogenesis using porcine coronary artery endothelial cells (PCAECs) and a mouse myocardial I/R model. Our data demonstrate that exposure of PCAECs to hypoxia for 2 h followed by 1 h of reoxygenation significantly increased ROS formation. Pretreatment with the NADPH oxidase inhibitors, diphenyleneiodonium (DPI, 10 microM) and apocynin (Apo, 200 and 600 microM), significantly attenuated H/R-induced ROS formation. Furthermore, exposure of PCAECs to H/R caused a significant increase in Akt and ERK1/2 activation. Exposure of PCAEC spheroids and mouse aortic rings to H/R significantly increased endothelial spheroid sprouting and vessel outgrowth, whereas pharmacological inhibition of NADPH oxidase or genetic deletion of the NADPH oxidase subunit, p47(phox) (p47(phox-/-)), significantly suppressed these changes. With the use of a mouse I/R model, our data further show that the increases in myocardial Akt and ERK1/2 activation and vascular endothelial growth factor (VEGF) expression were markedly blunted in the p47(phox-/-) mouse subjected to myocardial I/R compared with the wild-type mouse. Our findings underscore the important role of NADPH oxidase and its subunit p47(phox) in modulating Akt and ERK1/2 activation, angiogenic growth factor expression, and angiogenesis in myocardium undergoing I/R.