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Dimethylfumarate protects against TNF-α-induced secretion of inflammatory cytokines in human endothelial cells.

Gerhardt S, König V, Doll M, Hailemariam-Jahn T, Hrgovic I, Zöller N, Kaufmann R, Kippenberger S, Meissner M - J Inflamm (Lond) (2015)

Bottom Line: Furthermore, we found that DMF slightly inhibited the early degradation of IκBα.This action is regulated by reduced p65 activity and nuclear translocation, which can be explained in part by the reduced early degradation of IκBα and more important the reduced phosphorylation of p65 at Serine 536.These effects were independent of the p38, PI3K and p42/44 signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Venereology and Allergology, Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany ; Department of Cardiology, Gutenberg-University, Mainz, Germany.

ABSTRACT

Background: Inflammation, angiogenesis and oxidative stress have been implicated in the pathogenesis of various vascular diseases. Recent evidence suggests that dimethylfumarate (DMF), an antiposriatic and anti-multiple sclerosis agent, possesses anti-inflammatory, anti-oxidative and anti-angiogenic properties. Here, we analyze the influence of DMF on TNF-α-induced expression of the important pro-inflammatory and pro-atherogenic chemokine MCP-1 and investigate the underlying mechanisms of this expression.

Findings: We analyzed constitutive and TNF-α-induced expression of MCP-1 in human umbilical vascular endothelial cells (HUVEC) +/- DMF treatment via enzyme-linkes immunosorbent assay (ELISA). DMF significantly inhibited the protein expression levels in a time- and concentration-dependent manner. Furthermore, MCP-1 mRNA expression was also reduced in response to DMF, as demonstrated by RT-PCR. Thus, the regulation occurs at the transcriptional level. Interestingly, DMF prolonged the TNF-α-induced p38 and JNK phosphorylation in HUVEC, as demonstrated by Western blot analysis; however, the p38 and JNK inhibitor SB203580 did not affect the DMF-conveyed suppression of TNF-α-induced MCP-1 expression. DMF suppressed the TNF-α-induced nuclear translocation and phosphorylation (Serine 536) of p65 in these cells. These results were additionally approved by p65 luciferase promoter assays. Furthermore, we found that DMF slightly inhibited the early degradation of IκBα. In addition, we verified our results using other important inflammatory cytokines such as CCL-5, PDGF-BB, GM-CSF and IL-6.

Conclusion: DMF suppresses various TNF-α-induced pro-inflammatory and pro-atherogenic cytokines/chemokines in human endothelial cells. This action is regulated by reduced p65 activity and nuclear translocation, which can be explained in part by the reduced early degradation of IκBα and more important the reduced phosphorylation of p65 at Serine 536. These effects were independent of the p38, PI3K and p42/44 signaling pathways. As a result, DMF might be suitable for treating patients with vascular diseases.

No MeSH data available.


Related in: MedlinePlus

Analysis of the effects of DMF on p38, JNK, Akt and p42/44 phosphorylation in TNF-α-treated HUVECs and the influence on MCP-1 expression. a p38, JNK, Akt and p42/44 expression and phosphorylation: Western blot analysis of HUVECs treated with vehicle (solvent only), TNF-α (20 ng/ml) or DMF (80 μM, 3 h pre-treatment) + TNFα for 60 min. b Densitometry analysis: The results were normalized to the expression of the nonposphorylated controls (p38, JNK, AKT, p42/44). The relative expression of the posphorylated protein is presented in arbitrary units (arb. units). The mean values from at least three independent experiments are presented as the mean ± standard deviation. The TNF-α only treated bands served as control to the combinational treatment (DMF+ TNF-α). We analyzed theata using the Student’s t test. *p <0.05; n.s. not significant.c Signaling pathway blockade: MCP-1 ELISA of HUVECs treated with vehicle (solvent only), TNF-α (20 ng/ml) or DMF (80 μM, 3 h pre-treatment) + TNF-α for 60 min. We performed the blockade of the signaling pathways via treatment with SB203580 (1 μM), Wortmannin (5 μM) and PD 98059 (30 μM) 30 min before the main treatment. We present the mean values from three triplicate experiments as the mean ± SEM. We analyzed the data using the Student’s t-test. *p < 0.05 versus TNF-α; **p < 0.05 versus control
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Fig3: Analysis of the effects of DMF on p38, JNK, Akt and p42/44 phosphorylation in TNF-α-treated HUVECs and the influence on MCP-1 expression. a p38, JNK, Akt and p42/44 expression and phosphorylation: Western blot analysis of HUVECs treated with vehicle (solvent only), TNF-α (20 ng/ml) or DMF (80 μM, 3 h pre-treatment) + TNFα for 60 min. b Densitometry analysis: The results were normalized to the expression of the nonposphorylated controls (p38, JNK, AKT, p42/44). The relative expression of the posphorylated protein is presented in arbitrary units (arb. units). The mean values from at least three independent experiments are presented as the mean ± standard deviation. The TNF-α only treated bands served as control to the combinational treatment (DMF+ TNF-α). We analyzed theata using the Student’s t test. *p <0.05; n.s. not significant.c Signaling pathway blockade: MCP-1 ELISA of HUVECs treated with vehicle (solvent only), TNF-α (20 ng/ml) or DMF (80 μM, 3 h pre-treatment) + TNF-α for 60 min. We performed the blockade of the signaling pathways via treatment with SB203580 (1 μM), Wortmannin (5 μM) and PD 98059 (30 μM) 30 min before the main treatment. We present the mean values from three triplicate experiments as the mean ± SEM. We analyzed the data using the Student’s t-test. *p < 0.05 versus TNF-α; **p < 0.05 versus control

Mentions: The p38/JNK, PI3K/Akt and p42/44 signaling pathways are known to be important in TNF-α-induced MCP-1 expression. Therefore, we analyzed the phosphorylation status of p38, JNK, Akt and p42/44 in HUVEC over time (0–60 min.) in response to TNF-α (20 ng/ml) or TNF-α plus DMF (80 μM, 3 h pre-treatment). Surprisingly, we found that DMF prolonged the TNF-α-induced phosphorylation of p38 and JNK in HUVECs significantly based on Western blot and densitometry analyses (Fig. 3a,b). For Akt phosphorylation, we observed an early significant (0 and 5 min.) superinduction but no prolonged phosphorylation compared with TNF-α treatment. The posphorylation of p42/44 was not influenced by DMF treatment. To determine whether these signaling pathways were responsible for the effects of DMF on MCP-1 expression, we performed an enzyme-linked immunosorbent assay (ELISA) with the p38/JNK inhibitor SB203580, the PI3K/Akt inhibitor Wortmannin and the p42/44 inhibitor PD98059. Interestingly, blocking these signaling pathyways did not affect the DMF-induced suppression of MCP-1 expression (Fig. 3c).Fig. 3


Dimethylfumarate protects against TNF-α-induced secretion of inflammatory cytokines in human endothelial cells.

Gerhardt S, König V, Doll M, Hailemariam-Jahn T, Hrgovic I, Zöller N, Kaufmann R, Kippenberger S, Meissner M - J Inflamm (Lond) (2015)

Analysis of the effects of DMF on p38, JNK, Akt and p42/44 phosphorylation in TNF-α-treated HUVECs and the influence on MCP-1 expression. a p38, JNK, Akt and p42/44 expression and phosphorylation: Western blot analysis of HUVECs treated with vehicle (solvent only), TNF-α (20 ng/ml) or DMF (80 μM, 3 h pre-treatment) + TNFα for 60 min. b Densitometry analysis: The results were normalized to the expression of the nonposphorylated controls (p38, JNK, AKT, p42/44). The relative expression of the posphorylated protein is presented in arbitrary units (arb. units). The mean values from at least three independent experiments are presented as the mean ± standard deviation. The TNF-α only treated bands served as control to the combinational treatment (DMF+ TNF-α). We analyzed theata using the Student’s t test. *p <0.05; n.s. not significant.c Signaling pathway blockade: MCP-1 ELISA of HUVECs treated with vehicle (solvent only), TNF-α (20 ng/ml) or DMF (80 μM, 3 h pre-treatment) + TNF-α for 60 min. We performed the blockade of the signaling pathways via treatment with SB203580 (1 μM), Wortmannin (5 μM) and PD 98059 (30 μM) 30 min before the main treatment. We present the mean values from three triplicate experiments as the mean ± SEM. We analyzed the data using the Student’s t-test. *p < 0.05 versus TNF-α; **p < 0.05 versus control
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4525722&req=5

Fig3: Analysis of the effects of DMF on p38, JNK, Akt and p42/44 phosphorylation in TNF-α-treated HUVECs and the influence on MCP-1 expression. a p38, JNK, Akt and p42/44 expression and phosphorylation: Western blot analysis of HUVECs treated with vehicle (solvent only), TNF-α (20 ng/ml) or DMF (80 μM, 3 h pre-treatment) + TNFα for 60 min. b Densitometry analysis: The results were normalized to the expression of the nonposphorylated controls (p38, JNK, AKT, p42/44). The relative expression of the posphorylated protein is presented in arbitrary units (arb. units). The mean values from at least three independent experiments are presented as the mean ± standard deviation. The TNF-α only treated bands served as control to the combinational treatment (DMF+ TNF-α). We analyzed theata using the Student’s t test. *p <0.05; n.s. not significant.c Signaling pathway blockade: MCP-1 ELISA of HUVECs treated with vehicle (solvent only), TNF-α (20 ng/ml) or DMF (80 μM, 3 h pre-treatment) + TNF-α for 60 min. We performed the blockade of the signaling pathways via treatment with SB203580 (1 μM), Wortmannin (5 μM) and PD 98059 (30 μM) 30 min before the main treatment. We present the mean values from three triplicate experiments as the mean ± SEM. We analyzed the data using the Student’s t-test. *p < 0.05 versus TNF-α; **p < 0.05 versus control
Mentions: The p38/JNK, PI3K/Akt and p42/44 signaling pathways are known to be important in TNF-α-induced MCP-1 expression. Therefore, we analyzed the phosphorylation status of p38, JNK, Akt and p42/44 in HUVEC over time (0–60 min.) in response to TNF-α (20 ng/ml) or TNF-α plus DMF (80 μM, 3 h pre-treatment). Surprisingly, we found that DMF prolonged the TNF-α-induced phosphorylation of p38 and JNK in HUVECs significantly based on Western blot and densitometry analyses (Fig. 3a,b). For Akt phosphorylation, we observed an early significant (0 and 5 min.) superinduction but no prolonged phosphorylation compared with TNF-α treatment. The posphorylation of p42/44 was not influenced by DMF treatment. To determine whether these signaling pathways were responsible for the effects of DMF on MCP-1 expression, we performed an enzyme-linked immunosorbent assay (ELISA) with the p38/JNK inhibitor SB203580, the PI3K/Akt inhibitor Wortmannin and the p42/44 inhibitor PD98059. Interestingly, blocking these signaling pathyways did not affect the DMF-induced suppression of MCP-1 expression (Fig. 3c).Fig. 3

Bottom Line: Furthermore, we found that DMF slightly inhibited the early degradation of IκBα.This action is regulated by reduced p65 activity and nuclear translocation, which can be explained in part by the reduced early degradation of IκBα and more important the reduced phosphorylation of p65 at Serine 536.These effects were independent of the p38, PI3K and p42/44 signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Venereology and Allergology, Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany ; Department of Cardiology, Gutenberg-University, Mainz, Germany.

ABSTRACT

Background: Inflammation, angiogenesis and oxidative stress have been implicated in the pathogenesis of various vascular diseases. Recent evidence suggests that dimethylfumarate (DMF), an antiposriatic and anti-multiple sclerosis agent, possesses anti-inflammatory, anti-oxidative and anti-angiogenic properties. Here, we analyze the influence of DMF on TNF-α-induced expression of the important pro-inflammatory and pro-atherogenic chemokine MCP-1 and investigate the underlying mechanisms of this expression.

Findings: We analyzed constitutive and TNF-α-induced expression of MCP-1 in human umbilical vascular endothelial cells (HUVEC) +/- DMF treatment via enzyme-linkes immunosorbent assay (ELISA). DMF significantly inhibited the protein expression levels in a time- and concentration-dependent manner. Furthermore, MCP-1 mRNA expression was also reduced in response to DMF, as demonstrated by RT-PCR. Thus, the regulation occurs at the transcriptional level. Interestingly, DMF prolonged the TNF-α-induced p38 and JNK phosphorylation in HUVEC, as demonstrated by Western blot analysis; however, the p38 and JNK inhibitor SB203580 did not affect the DMF-conveyed suppression of TNF-α-induced MCP-1 expression. DMF suppressed the TNF-α-induced nuclear translocation and phosphorylation (Serine 536) of p65 in these cells. These results were additionally approved by p65 luciferase promoter assays. Furthermore, we found that DMF slightly inhibited the early degradation of IκBα. In addition, we verified our results using other important inflammatory cytokines such as CCL-5, PDGF-BB, GM-CSF and IL-6.

Conclusion: DMF suppresses various TNF-α-induced pro-inflammatory and pro-atherogenic cytokines/chemokines in human endothelial cells. This action is regulated by reduced p65 activity and nuclear translocation, which can be explained in part by the reduced early degradation of IκBα and more important the reduced phosphorylation of p65 at Serine 536. These effects were independent of the p38, PI3K and p42/44 signaling pathways. As a result, DMF might be suitable for treating patients with vascular diseases.

No MeSH data available.


Related in: MedlinePlus