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Foam cell-derived 4-hydroxynonenal induces endothelial cell senescence in a TXNIP-dependent manner.

Riahi Y, Kaiser N, Cohen G, Abd-Elrahman I, Blum G, Shapira OM, Koler T, Simionescu M, Sima AV, Zarkovic N, Zarkovic K, Orioli M, Aldini G, Cerasi E, Leibowitz G, Sasson S - J. Cell. Mol. Med. (2015)

Bottom Line: This senescent phenotype was mediated by 4-hydroxnonenal (4-HNE), a lipid peroxidation product secreted from foam cells; scavenging of 4-HNE in the co-culture medium blunted this effect.Molecular manipulation of TXNIP expression confirmed its involvement in foam cell-induced senescence.Collectively, these findings suggest that foam cell-released 4-HNE activates PPARδ in VEC, leading to increased TXNIP expression and consequently to senescence.

View Article: PubMed Central - PubMed

Affiliation: Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University, Jerusalem, Israel.

No MeSH data available.


Related in: MedlinePlus

PPARδ modulates the expression of TXNIP and senescence markers in VEC. (A) VEC were treated with 10 μM 4-HNE without (control) or with 1 μM GSK0660 (GSK) for 7 days with daily changes of medium. The expression of TXNIP and the senescence markers was determined in lysed cells by Western blot analysis. The level of TXNIP and the senescence markers in control lysates was taken as 100%. *P < 0.05 for differences from the respective controls (mean ± SEM, n = 3). (B) VEC cultures were maintained for 48 hrs without (control) or with the indicated agonists for PPARα, -γ and -δ. Cells were then taken for Western blot analysis of TXNIP expression. *P < 0.05, for differences from control cells treated with vehicle only. (C) VEC cultures were transfected with the following expression vectors: pSG5-hPPARα, pCMX-hPPARγ1, pCMX-hPPARγ2 or pCDNA-hPPARδ and co-transfected with pSVPORT-hRXR, pEGFP-N1 plasmid, 3xPPRE-TK-Luciferase reporter plasmid and Renilla luciferase plasmid. The cells were incubated for 48 hrs. During the last 24 hrs of incubation the cultures were incubated without (control) or with 1 or 10 μM 4-HNE. The cells were then harvested, lysed and the relative luciferase activity was determined and normalized as described under Materials and methods. *P < 0.05, for differences from the respective controls, taken as 1 unit (mean ± SEM, n = 3). (D) VEC cultures were co-transfected with a luciferase reporter construct containing the human TXNIP promoter and with the Renilla luciferase reporter plasmid, as described under Materials and methods. After 24 hrs of incubation the medium was changed and cells were incubated for additional 24 hrs without (control) or with 10 μM 4-HNE in the absence or presence of 1 μM GSK0660. Cells were then lysed and taken for the luciferase activity assay as described above. The relative luciferase activity was normalized to untreated control. *P < 0.05, for differences from the indicated groups (mean ± SEM, n = 3).
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fig05: PPARδ modulates the expression of TXNIP and senescence markers in VEC. (A) VEC were treated with 10 μM 4-HNE without (control) or with 1 μM GSK0660 (GSK) for 7 days with daily changes of medium. The expression of TXNIP and the senescence markers was determined in lysed cells by Western blot analysis. The level of TXNIP and the senescence markers in control lysates was taken as 100%. *P < 0.05 for differences from the respective controls (mean ± SEM, n = 3). (B) VEC cultures were maintained for 48 hrs without (control) or with the indicated agonists for PPARα, -γ and -δ. Cells were then taken for Western blot analysis of TXNIP expression. *P < 0.05, for differences from control cells treated with vehicle only. (C) VEC cultures were transfected with the following expression vectors: pSG5-hPPARα, pCMX-hPPARγ1, pCMX-hPPARγ2 or pCDNA-hPPARδ and co-transfected with pSVPORT-hRXR, pEGFP-N1 plasmid, 3xPPRE-TK-Luciferase reporter plasmid and Renilla luciferase plasmid. The cells were incubated for 48 hrs. During the last 24 hrs of incubation the cultures were incubated without (control) or with 1 or 10 μM 4-HNE. The cells were then harvested, lysed and the relative luciferase activity was determined and normalized as described under Materials and methods. *P < 0.05, for differences from the respective controls, taken as 1 unit (mean ± SEM, n = 3). (D) VEC cultures were co-transfected with a luciferase reporter construct containing the human TXNIP promoter and with the Renilla luciferase reporter plasmid, as described under Materials and methods. After 24 hrs of incubation the medium was changed and cells were incubated for additional 24 hrs without (control) or with 10 μM 4-HNE in the absence or presence of 1 μM GSK0660. Cells were then lysed and taken for the luciferase activity assay as described above. The relative luciferase activity was normalized to untreated control. *P < 0.05, for differences from the indicated groups (mean ± SEM, n = 3).

Mentions: Others and we have shown that 4-HNE activates PPARδ 42,43; hence we investigated whether this mechanism is also involved in 4-HNE-induced senescence in VEC. Figure5A shows that the selective PPARδ antagonist GSK0660 abolished 4-HNE effects on the expression of the different senescence markers and prevented the up-regulation of TXNIP. In addition, VEC were exposed for 48 hrs to selective agonists for the various PPAR receptors at their effective concentrations, as described by us before in the same VEC culture 44. The selective PPARδ agonist, GW501516, markedly augmented TXNIP expression, whereas, the PPARα agonist Wy14643 and the PPARγ agonist, troglitazone, did not modify significantly TXNIP expression (Fig.5B). The hypothesis that 4-HNE activates PPARδ in VEC was further investigated by assessing the transactivation of PPRE-coupled luciferase reporter 44. Briefly, VEC were co-transfected with the PPAR response element (PPRE)-luciferase reporter vector (3xPPRE-TK-Luciferase) and a combination of a specific human PPAR vector and RXR expression vector, followed by a 48 hrs treatment with 4-HNE (Fig.5C). A significant increase in luciferase activity in cells exposed to 10 μM 4-HNE was observed only in cells over-expressing the human PPARδ. The expression of PPARα was not altered in the presence of 4-HNE, whereas the expression of PPARγ1 and PPARγ2 was somewhat reduced. To further test whether PPARδ mediated the enhancement of TXNIP transcription by 4-HNE, VEC were transfected with the human TXNIP promoter (1777 bp upstream of the ATG start codon)-luciferase vector. TXNIP promoter activity was increased by 4-HNE and reduced in the presence of the PPARδ antagonist GSK0660 (Fig.5D).


Foam cell-derived 4-hydroxynonenal induces endothelial cell senescence in a TXNIP-dependent manner.

Riahi Y, Kaiser N, Cohen G, Abd-Elrahman I, Blum G, Shapira OM, Koler T, Simionescu M, Sima AV, Zarkovic N, Zarkovic K, Orioli M, Aldini G, Cerasi E, Leibowitz G, Sasson S - J. Cell. Mol. Med. (2015)

PPARδ modulates the expression of TXNIP and senescence markers in VEC. (A) VEC were treated with 10 μM 4-HNE without (control) or with 1 μM GSK0660 (GSK) for 7 days with daily changes of medium. The expression of TXNIP and the senescence markers was determined in lysed cells by Western blot analysis. The level of TXNIP and the senescence markers in control lysates was taken as 100%. *P < 0.05 for differences from the respective controls (mean ± SEM, n = 3). (B) VEC cultures were maintained for 48 hrs without (control) or with the indicated agonists for PPARα, -γ and -δ. Cells were then taken for Western blot analysis of TXNIP expression. *P < 0.05, for differences from control cells treated with vehicle only. (C) VEC cultures were transfected with the following expression vectors: pSG5-hPPARα, pCMX-hPPARγ1, pCMX-hPPARγ2 or pCDNA-hPPARδ and co-transfected with pSVPORT-hRXR, pEGFP-N1 plasmid, 3xPPRE-TK-Luciferase reporter plasmid and Renilla luciferase plasmid. The cells were incubated for 48 hrs. During the last 24 hrs of incubation the cultures were incubated without (control) or with 1 or 10 μM 4-HNE. The cells were then harvested, lysed and the relative luciferase activity was determined and normalized as described under Materials and methods. *P < 0.05, for differences from the respective controls, taken as 1 unit (mean ± SEM, n = 3). (D) VEC cultures were co-transfected with a luciferase reporter construct containing the human TXNIP promoter and with the Renilla luciferase reporter plasmid, as described under Materials and methods. After 24 hrs of incubation the medium was changed and cells were incubated for additional 24 hrs without (control) or with 10 μM 4-HNE in the absence or presence of 1 μM GSK0660. Cells were then lysed and taken for the luciferase activity assay as described above. The relative luciferase activity was normalized to untreated control. *P < 0.05, for differences from the indicated groups (mean ± SEM, n = 3).
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fig05: PPARδ modulates the expression of TXNIP and senescence markers in VEC. (A) VEC were treated with 10 μM 4-HNE without (control) or with 1 μM GSK0660 (GSK) for 7 days with daily changes of medium. The expression of TXNIP and the senescence markers was determined in lysed cells by Western blot analysis. The level of TXNIP and the senescence markers in control lysates was taken as 100%. *P < 0.05 for differences from the respective controls (mean ± SEM, n = 3). (B) VEC cultures were maintained for 48 hrs without (control) or with the indicated agonists for PPARα, -γ and -δ. Cells were then taken for Western blot analysis of TXNIP expression. *P < 0.05, for differences from control cells treated with vehicle only. (C) VEC cultures were transfected with the following expression vectors: pSG5-hPPARα, pCMX-hPPARγ1, pCMX-hPPARγ2 or pCDNA-hPPARδ and co-transfected with pSVPORT-hRXR, pEGFP-N1 plasmid, 3xPPRE-TK-Luciferase reporter plasmid and Renilla luciferase plasmid. The cells were incubated for 48 hrs. During the last 24 hrs of incubation the cultures were incubated without (control) or with 1 or 10 μM 4-HNE. The cells were then harvested, lysed and the relative luciferase activity was determined and normalized as described under Materials and methods. *P < 0.05, for differences from the respective controls, taken as 1 unit (mean ± SEM, n = 3). (D) VEC cultures were co-transfected with a luciferase reporter construct containing the human TXNIP promoter and with the Renilla luciferase reporter plasmid, as described under Materials and methods. After 24 hrs of incubation the medium was changed and cells were incubated for additional 24 hrs without (control) or with 10 μM 4-HNE in the absence or presence of 1 μM GSK0660. Cells were then lysed and taken for the luciferase activity assay as described above. The relative luciferase activity was normalized to untreated control. *P < 0.05, for differences from the indicated groups (mean ± SEM, n = 3).
Mentions: Others and we have shown that 4-HNE activates PPARδ 42,43; hence we investigated whether this mechanism is also involved in 4-HNE-induced senescence in VEC. Figure5A shows that the selective PPARδ antagonist GSK0660 abolished 4-HNE effects on the expression of the different senescence markers and prevented the up-regulation of TXNIP. In addition, VEC were exposed for 48 hrs to selective agonists for the various PPAR receptors at their effective concentrations, as described by us before in the same VEC culture 44. The selective PPARδ agonist, GW501516, markedly augmented TXNIP expression, whereas, the PPARα agonist Wy14643 and the PPARγ agonist, troglitazone, did not modify significantly TXNIP expression (Fig.5B). The hypothesis that 4-HNE activates PPARδ in VEC was further investigated by assessing the transactivation of PPRE-coupled luciferase reporter 44. Briefly, VEC were co-transfected with the PPAR response element (PPRE)-luciferase reporter vector (3xPPRE-TK-Luciferase) and a combination of a specific human PPAR vector and RXR expression vector, followed by a 48 hrs treatment with 4-HNE (Fig.5C). A significant increase in luciferase activity in cells exposed to 10 μM 4-HNE was observed only in cells over-expressing the human PPARδ. The expression of PPARα was not altered in the presence of 4-HNE, whereas the expression of PPARγ1 and PPARγ2 was somewhat reduced. To further test whether PPARδ mediated the enhancement of TXNIP transcription by 4-HNE, VEC were transfected with the human TXNIP promoter (1777 bp upstream of the ATG start codon)-luciferase vector. TXNIP promoter activity was increased by 4-HNE and reduced in the presence of the PPARδ antagonist GSK0660 (Fig.5D).

Bottom Line: This senescent phenotype was mediated by 4-hydroxnonenal (4-HNE), a lipid peroxidation product secreted from foam cells; scavenging of 4-HNE in the co-culture medium blunted this effect.Molecular manipulation of TXNIP expression confirmed its involvement in foam cell-induced senescence.Collectively, these findings suggest that foam cell-released 4-HNE activates PPARδ in VEC, leading to increased TXNIP expression and consequently to senescence.

View Article: PubMed Central - PubMed

Affiliation: Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University, Jerusalem, Israel.

No MeSH data available.


Related in: MedlinePlus