NF-kappaB Activation In Endothelial Cells Research Articles

NOVEL PATHOPHYSIOLOGICAL ROLES OF ALPHA-SYNUCLEIN (SNCA) IN AGE-RELATED VASCULAR ENDOTHELIAL DYSFUNCTION

Objective: Although SNCA is one of the well-known pathological molecule of synucleinopathy in neurons, its physiological roles remain to be identified. In the present study, we sought to elucidate the novel functions of SNCA in endothelium. Design and method: Analysis for human samples, in vitro experiments using endothelial cells (ECs), and in vivo investigation using SNCA knockout or SNCA/ApoE double knockout mice were performed. Results: We found SNCA was expressed in and secreted from ECs by functional screening for anti-inflammatory molecules and detectable in circulating blood. Intriguingly, the human population study revealed serum SNCA levels decreased with aging and displayed inverse correlation with blood pressure and insulin sensitivity, which indicated protective properties of circulating SNCA on vascular endothelial function. Furthermore, we also found SNCA knockout mice displayed phenotypes of metabolic syndrome such as hypertension, impaired glucose metabolism, and dyslipidemia. Based on these preliminary data, we sought to elucidate the physiological functions of endogenous and exogenous SNCA for ECs. Exogenous treatment with recombinant SNCA (rSNCA) promoted eNOS activation and nitric oxide production via Gab1/PI3K/Akt pathway in ECs, followed by cGMP production in co-cultured vascular smooth muscle cells. Treatment with rSNCA also suppressed TNF-alpha induced NF-kappaB activation in ECs. As to endogenous SNCA, replicative senescence showed attenuation of SNCA expression in ECs and siRNA-mediated silencing of SNCA induced eNOS inactivation and cell senescence assessed by beta-gal activity along with decreased Sirt1 expression and increased p53 expression. SNCA overexpression displayed NF-kappaB inactivation in ECs. In ex vivo study, aortas from SNCA knockout mice showed impairment of acetylcholine-induced relaxation possibly due to eNOS dysfunction. In in vivo study utilizing atherosclerosis model of SNCA/ApoE double-knockout mice showed exaggerated expression of inflammatory genes which play important roles in atherogenesis. Conclusions: In conclusion, these results indicate exogenous and endogenous SNCA in ECs might physiologically maintain cerebral vascular integrity. Aging or aggregation-related loss-of-function of SNCA in ECs might be partially correlated with age-related hypertension or clinical features of dementia with Lewy body such as fluctuating cognitive function and marked sensitivity of antipsychotic which could be modified by cerebral blood flow and vascular permeability.

Ca2+ influx via TRPC channels induces NF-κB-dependent A20 expression to prevent thrombin-induced apoptosis in endothelial cells

NF-kappaB signaling is known to induce the expression of antiapoptotic and proinflammatory genes in endothelial cells (ECs). We have shown recently that Ca(2+) influx through canonical transient receptor potential (TRPC) channels activates NF-kappaB in ECs. Here we show that Ca(2+) influx signal prevents thrombin-induced apoptosis by inducing NF-kappaB-dependent A20 expression in ECs. Knockdown of TRPC1 expressed in human umbilical vein ECs with small interfering RNA (siRNA) suppressed thrombin-induced Ca(2+) influx and NF-kappaB activation in ECs. Interestingly, we observed that thrombin induced >25% of cell death (apoptosis) in TRPC1-knockdown ECs whereas thrombin had no effect on control or control siRNA-transfected ECs. To understand the basis of EC survival, we performed gene microarray analysis using ECs. Thrombin stimulation increased only a set of NF-kappaB-regulated genes 3- to 14-fold over basal levels in ECs. Expression of the antiapoptotic gene A20 was the highest among these upregulated genes. Like TRPC1 knockdown, thrombin induced apoptosis in A20-knockdown ECs. To address the importance of Ca(2+) influx signal, we measured thrombin-induced A20 expression in control and TRPC1-knockdown ECs. Thrombin-induced p65/RelA binding to A20 promoter-specific NF-kappaB sequence and A20 protein expression were suppressed in TRPC1-knockdown ECs compared with control ECs. Furthermore, in TRPC1-knockdown ECs, thrombin induced the expression of proapoptotic proteins caspase-3 and BAX. Importantly, thrombin-induced apoptosis in TRPC1-knockdown ECs was prevented by adenovirus-mediated expression of A20. These results suggest that Ca(2+) influx via TRPC channels plays a critical role in the mechanism of cell survival signaling through A20 expression in ECs.

Sensitization of endothelial cells to VEGI-induced apoptosis by inhibiting the NF-κB pathway

Vascular endothelial growth inhibitor (VEGI) is an endogenous inhibitor of endothelial cell growth and a promising candidate for cancer therapy. VEGI is able to inhibit tumor growth by specifically targeting the tumor neovasculature. Increasing the anti-angiogenic potential of this cytokine is of great interest for its therapeutic potential. NF-kappaB is known to have an integral role in TNF superfamily signaling, acting as a pro-survival factor. A role of VEGI-induced NF-kappaB activation in endothelial cells has yet to be described. Here we show that suppression of the NF-kappaB pathway can increase the apoptotic potential of VEGI. We used siRNA to deplete NF-kappaB or its activator IKK2 from adult bovine aortic endothelial cells. The siRNA treatments diminished VEGI-induced NF-kappaB activation, evidenced from a reduced extent of NF-kappaB nuclear translocation and diminished expression of NF-kappaB-target genes such as interleukins-6 and -1beta. The siRNA-treated endothelial cells when exposed to VEGI exhibited a marked decrease in cell viability and a significant increase in apoptosis. These results confirm that VEGI utilizes NF-kappaB as a pro-survival role factor in endothelial cells. We then examined whether a combination of VEGI with NF-kappaB inhibitors would constitute a more potential therapeutic regiment. We found that in the presence of the NF-kappaB inhibitors curcumin or BMS-345541 there was a marked increase in the apoptotic potential of VEGI on endothelial cells. These findings indicate that a combination therapy using VEGI and NF-kappaB inhibitors could be a potent approach for cancer treatment.

NF-kappaB activity in endothelial cells is modulated by cell substratum interactions and influences chemokine-mediated adhesion of natural killer cells.

Because changes in subendothelial matrix composition are associated with alterations of the endothelial immune phenotype, we sought to understand if cytokine-induced NF-kappaB activity and downstream effects depend on substrate adherence of endothelial cells (EC). We compared the upstream phosphorylation cascade, activation of NF-kappaB, and expression/secretion of downstream effects of EC grown on tissue culture polystyrene plates (TCPS) with EC embedded within collagen-based matrices (MEEC). Adhesion of natural killer (NK) cells was quantified in vitro and in vivo. NF-kappaB subunit p65 nuclear levels were significantly lower and p50 significantly higher in cytokine-stimulated MEEC than in EC-TCPS. Despite similar surface expression of TNF-alpha receptors, MEEC had significantly decreased secretion and expression of IL-6, IL-8, MCP-1, VCAM-1, and ICAM-1. Attenuated fractalkine expression and secretion in MEEC (two to threefold lower than in EC-TCPS; p < 0.0002) correlated with 3.7-fold lower NK cell adhesion to EC (6,335 +/- 420 vs. 1,735 +/- 135 cpm; p < 0.0002). Furthermore, NK cell infiltration into sites of EC implantation in vivo was significantly reduced when EC were embedded within matrix. Matrix embedding enables control of EC substratum interaction. This in turn regulates chemokine and surface molecule expression and secretion, in particular of those compounds within NF-kappaB pathways, chemoattraction of NK cells, local inflammation, and tissue repair.

Hyperhomocysteinemia activates nuclear factor-kappaB in endothelial cells via oxidative stress.

Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases. Our previous studies demonstrated an important interaction between nuclear factor-kappaB (NF-kappaB) activation and homocysteine (Hcy)-induced chemokine expression in vascular smooth muscle cells and macrophages. The objective of the present study was to investigate the in vivo effect of hyperhomocysteinemia on NF-kappaB activation and the underlying mechanism of Hcy-induced NF-kappaB activation in endothelial cells. Hyperhomocysteinemia was induced in Sprague-Dawley rats after 4 weeks of a high-methionine diet. The activated form of NF-kappaB and increased level of superoxide anions were detected in the endothelium of aortas isolated from hyperhomocysteinemic rats. The underlying mechanism of Hcy-induced NF-kappaB activation was investigated in human umbilical cord vein endothelial cells and in human aortic endothelial cells. Incubation of cells with Hcy (100 micromol/L) activated IkappaB kinases (IKKalpha and IKKbeta), leading to phosphorylation and subsequent degradation of IkappaBalpha. As a consequence, NF-kappaB nuclear translocation, enhanced NF-kappaB/DNA binding activity, and increased transcriptional activity occurred. Additional analysis revealed a marked elevation of superoxide anion levels in Hcy-treated cells. Treatment of cells with a superoxide anion scavenger (polyethylene glycol-superoxide dismutase) or IkappaB kinase inhibitor (prostaglandin A(1)) could prevent Hcy-induced activation of IKK kinases and NF-kappaB in endothelial cells. In conclusion, these results suggest that Hcy-induced superoxide anion production may play a potential role for NF-kappaB activation in the early stages of atherosclerosis in the vascular wall via activation of IkappaB kinases.

Inhibition of Tumor Necrosis Factor α-mediated NFκB Activation and Leukocyte Adhesion, with Enhanced Endothelial Apoptosis, by G Protein-linked Receptor (TP) Ligands

Tumor necrosis factor (TNF) alpha is a critical mediator of inflammation; however, TNFalpha is rarely released alone and the "cross-talk" between different classes of inflammatory mediators is largely unexplored. Thromboxane A(2) (TXA(2)) is released during I/R injury and exerts its effects via a G protein-linked receptor (TP). In this study, we found that TXA(2) mimetics stimulate leukocyte adhesion molecule (LAM) expression on endothelium via TPbeta. The potential interaction between TXA(2) and TNFalpha in altering endothelial survival and LAM expression was examined. IBOP, a TXA(2) mimetic, attenuated TNFalpha-induced LAM expression in vitro, in a concentration-dependent manner, by preventing TNFalpha-enhanced gene expression, and also reduced TNFalpha-induced leukocyte adhesion to endothelium both in vitro and in vivo. IBOP abrogated TNFalpha-induced NFkappaB activation in endothelial cells, as determined by reduced IkappaB phosphorylation and NFkappaB nuclear translocation, by inhibiting the assembly of signaling intermediates with the intracellular domain of TNF receptors 1 and 2 in response to TNFalpha. This inhibition resulted from the Galpha(q)-mediated enhancement of STAT1 activation and was reversed by anti-STAT1 antisense oligonucleotides. TNFalpha-mediated TNFR1-FADD association and caspase 8 activation were not inhibited by IBOP co-stimulation, however, resulting in a 2.6-fold increase in endothelial cell apoptosis. By stimulating the vessel wall and inducing endothelial cell apoptosis, TXA(2), in combination with TNFalpha, may hamper the angiogenic response during inflammation or ischemia, thus reducing revascularization and tissue viability.

NFkappaB decoy oligodeoxynucleotides reduce monocyte infiltration in renal allografts.

Monocyte influx secondary to ischemia-reperfusion conditions the renal allograft to rejection by presentation of antigens and production of cytokines. Monocyte influx depends on NFkappaB-dependent transcription of genes encoding adhesion molecules and chemokines. Here we demonstrate that cationic liposomes containing phosphorothioated oligodeoxynucleotides (ODN) with the kappaB binding site serving as competitive binding decoy, can prevent TNF-alpha-induced NFkappaB activity in endothelial cells in vitro. In an allogenic rat kidney transplantation model (BN to LEW), we show that perfusing the renal allograft with this decoy prior to transplantation abolishes nuclear NFkappaB activity in vivo and inhibits VCAM-1 expression in the donor endothelium (P<0.05). At 24 h postreperfusion, periarterial infiltration of monocytes/macrophages was significantly reduced in decoy ODN-treated allografts compared to control allografts (3.7+/-0.7 vs. 9.2+/-1.2 macrophages/vessel; P<0.01). At 72 h, there was a reduction of tubulointerstitial macrophage infiltration in decoy ODN-treated kidneys compared to controls (75.6+/-13.9 vs. 120.0+/-11.2 macrophages/tubulointerstitial area; P<0.05). In conclusion, perfusion of the renal allograft with NFkappaB decoy ODN prior to transplantation decreases the initial inflammatory response in a stringent, nonimmunosuppressed allogenic transplantation model. Therefore, the NFkappaB decoy approach may be useful to explore the role of endothelium and macrophages in graft rejection and may be developed into a graft-specific immunosuppressive strategy allowing reduction of systemic immunosuppression on organ transplantation.

Oxidative stress induces NF-kappaB nuclear translocation without degradation of IkappaBalpha.

Rel/NF-kappaB, an oxidative stress-responsive transcription factor, participates transiently in the control of gene expression. The cellular mechanisms that mediate NF-kappaB activation during ischemia (and during reperfusion in the course of treating ischemia) are not known. To investigate the NF-kappaB activation induced during oxidative stress, we examined human cardiac tissue obtained during surgical procedures requiring cardiopulmonary bypass. In vitro, we examined human umbilical vein endothelial cells (HUVECs) exposed to hypoxia, reoxygenation after hypoxia, or a reactive oxygen intermediate (H(2)O(2)). Electrophoretic mobility shift assays performed on right atrial tissue revealed prominent NF-kappaB activation after hearts had been exposed to ischemia and reperfusion. The assays also showed that NF-kappaB activation was observed in hypoxic HUVECs after reoxygenation and in cultures treated with H(2)O(2) (500 micromol/L). Pervanadate (200 micromol/L) also induced marked NF-kappaB activation in HUVECs, indicating that H(2)O(2)-induced NF-kappaB activation is potentiated by the inhibition of tyrosine phosphatases. Western blotting of cytoplasmic IkappaBalpha demonstrated that NF-kappaB activation induced by oxidative stress was not associated with IkappaBalpha degradation. In contrast, tumor necrosis factor-alpha-induced NF-kappaB activation occurred in concert with degradation of IkappaBalpha. Inhibition of IkappaBalpha degradation with a proteasome inhibitor, MG-115, blocked NF-kappaB activation induced by tumor necrosis factor-alpha; however, MG-115 had no effect on NF-kappaB activation during oxidative stress. This study demonstrated a stimulus-specific mechanism of NF-kappaB activation in endothelial cells that acts independently of IkappaBalpha degradation and may require tyrosine phosphorylation.

Suppressive Effects of Anti-inflammatory Agents on Human Endothelial Cell Activation and Induction of Heat Shock Proteins

Studies from our laboratory have shown that the earliest stages of atherosclerosis may be mediated by an autoimmune reaction against heat shock protein 60 (Hsp60). The interactions of Hsp60-specific T cells with arterial endothelial cells (EC) require expression of both Hsp60 and certain adhesion molecules shown to be induced simultaneously in EC by mechanical and other types of stress. Recently, it was shown that suppression of T cell-mediated immune responses by cyclosporin A (CyA) enhanced atherosclerotic lesion formation in mice. In contrast, aspirin was found to lower the risk of myocardial infarction in men. These conflicting observations may be due to different effects of anti-inflammatory agents on adhesion molecule and Hsp expression in EC, respectively. In the present study, we analyzed the effects of CyA, aspirin, and indomethacin on T cell proliferation using a proliferation assay. To explore the expression of adhesion molecules, monocyte chemoattractant protein-1 (MCP-1), and Hsp60 in human umbilical vein endothelial cells (HUVECs), Northern blot analyses were used. To examine the activation status of the transcription factors nuclear factor kappaB (NF-kappaB) and heat shock factor-1 (HSF-1), electrophoretic mobility shift assays were performed. With the exception of indomethacin, the used immunosuppressive and anti-inflammatory agents significantly inhibited T cell proliferation in response to influenza virus antigen in a dose-dependent manner. Interestingly, CyA and indomethacin did not suppress tumor necrosis factor-alpha (TNF-alpha)-induced adhesion molecule expression on HUVECs, whereas aspirin had an inhibitory effect. These observations correlated with the modulation of NF-kappaB activity in EC. All agents tested induced expression of Hsp60 6 hr after application. In addition, aspirin and indomethacin, but not CyA, induced Hsp70 expression in HUVECs that correlated with induction of HSF-1 activity. Our results show that the tested agents (except indomethacin) are inhibitors of the T cell-mediated immune response, as expected, that aspirin is an effective suppressor of adhesion molecule expression, and that all three agents can induce Hsp60 in HUVECs. These data provide the molecular basis for the notion that (1) part of the anti-atherogenic effect of aspirin may be due to the prevention of the adhesion of sensitized T cells to stressed EC; (2) that part of the atherosclerosis-promoting effect of CyA may be due to its potential as an inducer of Hsp60 expression and its inability to down-regulate adhesion molecule expression on EC; and (3) that down-regulation of MCP-1 expression by aspirin may result in decreased recruitment of monocytes into the arterial intima beneath stressed EC.

Biphasic regulation of transcription factor nuclear factor-kappaB activity in human endothelial cells by lysophosphatidylcholine through protein kinase C-mediated pathway.

Lysophosphatidylcholine (lysoPC), which is generated in oxidized LDL (Ox-LDL) and abundantly exists in atherosclerotic arterial walls, has been shown to alter various endothelial functions and induces several endothelial genes expressed in atherosclerotic arterial walls. Nuclear factor-kappa B (NF-kappaB), a pleiotropic transcription factor, plays an important role in regulation of expression of various genes implicated in atherosclerosis. We have previously reported that lysoPC transferred from Ox-LDL to endothelial surface membrane activates endothelial protein kinase C (PKC), leading to modulated endothelial functions. This study was aimed at determining whether lysoPC could modulate activity of transcription factors in cultured human umbilical vein endothelial cells (HUVECs) by using electrophoretic mobility shift assay. LysoPC was found to increase DNA-binding activity of NF-kappaB in HUVECs within 15 minutes, which peaked at 1 to 2 hours and subsequently declined to the baseline level at 6 hours. Lower concentrations (5 to 15 micromol/L) of lysoPC markedly increased NF-kappaB activity, but higher concentration (50 micromol/L) of lysoPC inhibited the activity. Phorbol 12-myristate 13-acetate, a potent activator of PKC, also augmented NF-kappaB activity in HUVECs, mimicking the effects of lysoPC; furthermore, calphostin C and chelerythrine chloride, specific PKC inhibitors, and alpha-tocopherol, a clinically potent PKC inhibitor, suppressed the lysoPC-induced NF-kappaB activation. These results indicate that lysoPC regulates NF-kappaB activity in a biphasic manner dependent on its concentrations and incubation time in human endothelial cells and the endothelial PKC activation may in part be involved in the lysoPC-induced NF-kappaB activation. Thus, the time course and the positive and negative biphasic regulatory actions of lysoPC on NF-kappaB activity in endothelial cells might exhibit a unique effect of lysoPC in arterial walls on the different stages of atherosclerosis.

Lipid peroxidation is involved in the activation of NF-kappaB by tumor necrosis factor but not interleukin-1 in the human endothelial cell line ECV304. Lack of involvement of H2O2 in NF-kappaB activation by either cytokine in both primary and transformed endothelial cells.

It has been proposed that reactive oxygen species, and in particular H2O2, may be involved in the activation of NF-kappaB by diverse stimuli in different cell types. Here we have investigated the effect of a range of putative antioxidants on NF-kappaB activation by interleukin-1 and tumor necrosis factor as well as the ability of H2O2 to activate NF-kappaB in primary human umbilical vein endothelial cells and the transformed human endothelial cell line ECV304. Activation of NF-kappaB and stimulation of IkappaBalpha degradation by H2O2 was only evident in the transformed cells and required much longer contact times than that observed with interleukin-1 or tumor necrosis factor. Furthermore, only H2O2 was sensitive to N-acetyl-L-cysteine, and no increase in H2O2 was detected in response to either cytokine. Pyrrolidine dithiocarbamate has been purported to be a specific antioxidant inhibitor of NF-kappaB that acts independently of activating agent or cell type. However, we found that tumor necrosis factor- but not interleukin-1-driven NF-kappaB activation and IkappaBalpha degradation were sensitive to pyrrolidine dithiocarbamate in transformed cells, while neither pathway was inhibited in primary cells. Phorbol ester-mediated activation was sensitive in both transformed and primary cells. Other antioxidants failed to inhibit either cytokine, while the iron chelators desferrioxamine and 2,2,6, 6-tetramethylpiperidine-1-oxyl mimicked the pattern of inhibition seen for the dithiocarbamate. This suggested that pyrrolidine dithiocarbamate was inhibiting NF-kappaB activation in endothelial cells primarily through its iron-chelating properties. Tumor necrosis factor, but not interleukin-1, was found to induce lipid peroxidation in ECV304 cells. This was inhibited by pyrrolidine dithiocarbamate and desferrioxamine. t-Butyl hydroperoxide, which induces lipid peroxidation, activated NF-kappaB. Finally, butylated hydroxyanisole, which inhibits lipid peroxidation but has no iron-chelating properties, inhibited NF-kappaB activation by tumor necrosis factor but not interleukin-1. Taken together, the results argue against a role for H2O2 in NF-kappaB activation by cytokines in endothelial cells. Furthermore, tumor necrosis factor and interleukin-1 activate NF-kappaB through different mechanisms in ECV304 cells, with the tumor necrosis factor pathway involving iron-catalyzed lipid peroxidation.

Glucocorticoid-mediated Repression of NFκB Activity in Endothelial Cells Does Not Involve Induction of IκBα Synthesis

Repression of NFkappaB-dependent gene expression is one of the major elements of immunosuppression by glucocorticoids. Protein-protein interactions between the glucocorticoid receptor and NFkappaB have been characterized and shown to be a possible mechanism of mutual inhibition of transactivation properties. More recently, glucocorticoid-mediated induction of IkappaBalpha, an inhibitor of NFkappaB, has been described in monocytes and lymphocytes; an increase in IkappaBalpha mRNA and protein resulted in inactivation and cytosolic retention of NFkappaB. Thus, rather than the physical interaction between the glucocorticoid receptor and NFkappaB, the up-regulation of IkappaBalpha was presented as the key element in immunosuppression by glucocorticoids. In contrast, we show that the IkappaBalpha pathway is not involved in glucocorticoid-mediated inhibition of NFkappaB activity in endothelial cells. Although transcriptional activation by NFkappaB was significantly reduced in the presence of glucocorticoids, we did not detect induction of IkappaBalpha protein that could prevent nuclear translocation of NFkappaB upon stimulation with lipopolysaccharide or tumor necrosis factor alpha. Furthermore, treatment with glucocorticoids did not seem to affect the transcription rate or mRNA stability of IkappaBalpha. We therefore conclude that, although induction of IkappaBalpha expression by glucocorticoids seems to be of importance in monocytes and lymphocytes, it cannot explain inhibition of NFkappaB-dependent gene expression in endothelial cells. Our results emphasize the relevance of physical interaction between the glucocorticoid receptor and NFkappaB in endothelial cells and thus in suppression of inflammation by glucocorticoids.