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Inhibition of autophagy rescues palmitic acid-induced necroptosis of endothelial cells.

Khan MJ, Rizwan Alam M, Waldeck-Weiermair M, Karsten F, Groschner L, Riederer M, Hallström S, Rockenfeller P, Konya V, Heinemann A, Madeo F, Graier WF, Malli R - J. Biol. Chem. (2012)

Bottom Line: Here, we show that PA triggers autophagy, which did not counteract but in contrast promoted endothelial cell death.Moreover, the initiation of autophagy and cell death by PA was reduced in endothelial cells loaded with the Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-(acetoxymethyl) ester (BAPTA-AM), indicating that Ca(2+) triggers the fatal signaling of PA.In summary, we introduce an unexpected mechanism of lipotoxicity in endothelial cells and provide several novel strategies to counteract the lipotoxic signaling of PA.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, 8010 Graz, Austria.

ABSTRACT
Accumulation of palmitic acid (PA) in cells from nonadipose tissues is known to induce lipotoxicity resulting in cellular dysfunction and death. The exact molecular pathways of PA-induced cell death are still mysterious. Here, we show that PA triggers autophagy, which did not counteract but in contrast promoted endothelial cell death. The PA-induced cell death was predominantly necrotic as indicated by annexin V and propidium iodide (PI) staining, absence of caspase activity, low levels of DNA hypoploidy, and an early ATP depletion. In addition PA induced a strong elevation of mRNA levels of ubiquitin carboxyl-terminal hydrolase (CYLD), a known mediator of necroptosis. Moreover, siRNA-mediated knockdown of CYLD significantly antagonized PA-induced necrosis of endothelial cells. In contrast, inhibition and knockdown of receptor interacting protein kinase 1 (RIPK1) had no effect on PA-induced necrosis, indicating the induction of a CYLD-dependent but RIPK1-independent cell death pathway. PA was recognized as a strong and early inducer of autophagy. The inhibition of autophagy by both pharmacological inhibitors and genetic knockdown of the autophagy-specific genes, vacuolar protein sorting 34 (VPS34), and autophagy-related protein 7 (ATG7), could rescue the PA-induced death of endothelial cells. Moreover, the initiation of autophagy and cell death by PA was reduced in endothelial cells loaded with the Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-(acetoxymethyl) ester (BAPTA-AM), indicating that Ca(2+) triggers the fatal signaling of PA. In summary, we introduce an unexpected mechanism of lipotoxicity in endothelial cells and provide several novel strategies to counteract the lipotoxic signaling of PA.

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PA induces necrotic cell death in endothelial cells.A, cells were treated with BSA alone (white columns, n = 3), 0.5 mm OA (gray columns, n = 3), or 0.5 mm PA (black columns, n = 3) and cell viability was measured with MTT assay at the time points indicated. Fatty acids were complexed to BSA. Data were normalized to BSA as control and represented as percentage viability. *, p < 0.05 versus BSA. B, annexin V/PI costaining for the cells exposed to 0.5 mm PA (n = 4) or BSA alone (n = 4) (asterisk refers to annexin V/PI). *, p < 0.05 versus BSA. C, representative images of annexin V/PI-stained endothelial cells treated with 0.5 mm PA for 12 h (n = 4). Images were taken using a ×40 objective. D, cells were treated with the solvent (DMSO, control, -Z-VAD-fmk, n = 3) or with 20 μm Z-VAD-fmk (+zVAD-fmk, n = 3) prior to treatment with 0.5 mm PA or BSA alone and cellular viability was measured with MTT assay. *, p < 0.05 versus BSA (51.3 ± 6.7% in the Z-VAD-fmk treated as compared with 55.9 ± 7.7% in controls). E, caspase-3 activity measured with the FRET-based sensor Casper 3-GR, expressed as ratio GFP/FRET of randomly selected cells under control conditions (BSA, white column, n = 3, 81 cells) and after cell treatment with 0.5 mm PA for 16–18 h (n = 3, 102 cells). F, histograms of cell cycle analysis by flow cytometry for DNA hypoploidy. Cells were treated with BSA or 0.5 mm PA and were analyzed after 18 h of incubation. G, statistical analysis of the histograms shown in panel F. H, statistical data representing nmol of ATP/mg of protein in the cells exposed to 0.5 mm PA (black circles, dotted line, n = 6 for each time point) or BSA alone (white circles, continuous line, n = 6 for each time point). *, p < 0.05 versus BSA.
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Figure 1: PA induces necrotic cell death in endothelial cells.A, cells were treated with BSA alone (white columns, n = 3), 0.5 mm OA (gray columns, n = 3), or 0.5 mm PA (black columns, n = 3) and cell viability was measured with MTT assay at the time points indicated. Fatty acids were complexed to BSA. Data were normalized to BSA as control and represented as percentage viability. *, p < 0.05 versus BSA. B, annexin V/PI costaining for the cells exposed to 0.5 mm PA (n = 4) or BSA alone (n = 4) (asterisk refers to annexin V/PI). *, p < 0.05 versus BSA. C, representative images of annexin V/PI-stained endothelial cells treated with 0.5 mm PA for 12 h (n = 4). Images were taken using a ×40 objective. D, cells were treated with the solvent (DMSO, control, -Z-VAD-fmk, n = 3) or with 20 μm Z-VAD-fmk (+zVAD-fmk, n = 3) prior to treatment with 0.5 mm PA or BSA alone and cellular viability was measured with MTT assay. *, p < 0.05 versus BSA (51.3 ± 6.7% in the Z-VAD-fmk treated as compared with 55.9 ± 7.7% in controls). E, caspase-3 activity measured with the FRET-based sensor Casper 3-GR, expressed as ratio GFP/FRET of randomly selected cells under control conditions (BSA, white column, n = 3, 81 cells) and after cell treatment with 0.5 mm PA for 16–18 h (n = 3, 102 cells). F, histograms of cell cycle analysis by flow cytometry for DNA hypoploidy. Cells were treated with BSA or 0.5 mm PA and were analyzed after 18 h of incubation. G, statistical analysis of the histograms shown in panel F. H, statistical data representing nmol of ATP/mg of protein in the cells exposed to 0.5 mm PA (black circles, dotted line, n = 6 for each time point) or BSA alone (white circles, continuous line, n = 6 for each time point). *, p < 0.05 versus BSA.

Mentions: First we tested the susceptibility of the endothelial cell line, EA.hy926, to PA-induced cell death. For this purpose cells were treated with a complex of PA and BSA and cell viability was measured with the MTT assay at different times of incubation (Fig. 1A). After 12 h of incubation with PA, cell viability was found to be already significantly reduced and further decreased with time. The cell viability declined more than 80% after 28 h of incubation with PA (Fig. 1A), pointing to a strong cytotoxic effect of this saturated fatty acid on endothelial cells. The PA-induced cell death was further examined using long-term phase-contrast imaging, which showed an impaired cell proliferation and an accumulation of detached, dying cells upon incubation with PA (supplemental Fig. S1 and Movie S1). In contrast, cell treatment with OA did not affect the viability of the cells (Fig. 1A). These findings are in line with earlier studies using other cell types (26) and approved EA.hy926 cells as a suitable model to explore the molecular mechanisms of PA-mediated lipotoxicity.


Inhibition of autophagy rescues palmitic acid-induced necroptosis of endothelial cells.

Khan MJ, Rizwan Alam M, Waldeck-Weiermair M, Karsten F, Groschner L, Riederer M, Hallström S, Rockenfeller P, Konya V, Heinemann A, Madeo F, Graier WF, Malli R - J. Biol. Chem. (2012)

PA induces necrotic cell death in endothelial cells.A, cells were treated with BSA alone (white columns, n = 3), 0.5 mm OA (gray columns, n = 3), or 0.5 mm PA (black columns, n = 3) and cell viability was measured with MTT assay at the time points indicated. Fatty acids were complexed to BSA. Data were normalized to BSA as control and represented as percentage viability. *, p < 0.05 versus BSA. B, annexin V/PI costaining for the cells exposed to 0.5 mm PA (n = 4) or BSA alone (n = 4) (asterisk refers to annexin V/PI). *, p < 0.05 versus BSA. C, representative images of annexin V/PI-stained endothelial cells treated with 0.5 mm PA for 12 h (n = 4). Images were taken using a ×40 objective. D, cells were treated with the solvent (DMSO, control, -Z-VAD-fmk, n = 3) or with 20 μm Z-VAD-fmk (+zVAD-fmk, n = 3) prior to treatment with 0.5 mm PA or BSA alone and cellular viability was measured with MTT assay. *, p < 0.05 versus BSA (51.3 ± 6.7% in the Z-VAD-fmk treated as compared with 55.9 ± 7.7% in controls). E, caspase-3 activity measured with the FRET-based sensor Casper 3-GR, expressed as ratio GFP/FRET of randomly selected cells under control conditions (BSA, white column, n = 3, 81 cells) and after cell treatment with 0.5 mm PA for 16–18 h (n = 3, 102 cells). F, histograms of cell cycle analysis by flow cytometry for DNA hypoploidy. Cells were treated with BSA or 0.5 mm PA and were analyzed after 18 h of incubation. G, statistical analysis of the histograms shown in panel F. H, statistical data representing nmol of ATP/mg of protein in the cells exposed to 0.5 mm PA (black circles, dotted line, n = 6 for each time point) or BSA alone (white circles, continuous line, n = 6 for each time point). *, p < 0.05 versus BSA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3375534&req=5

Figure 1: PA induces necrotic cell death in endothelial cells.A, cells were treated with BSA alone (white columns, n = 3), 0.5 mm OA (gray columns, n = 3), or 0.5 mm PA (black columns, n = 3) and cell viability was measured with MTT assay at the time points indicated. Fatty acids were complexed to BSA. Data were normalized to BSA as control and represented as percentage viability. *, p < 0.05 versus BSA. B, annexin V/PI costaining for the cells exposed to 0.5 mm PA (n = 4) or BSA alone (n = 4) (asterisk refers to annexin V/PI). *, p < 0.05 versus BSA. C, representative images of annexin V/PI-stained endothelial cells treated with 0.5 mm PA for 12 h (n = 4). Images were taken using a ×40 objective. D, cells were treated with the solvent (DMSO, control, -Z-VAD-fmk, n = 3) or with 20 μm Z-VAD-fmk (+zVAD-fmk, n = 3) prior to treatment with 0.5 mm PA or BSA alone and cellular viability was measured with MTT assay. *, p < 0.05 versus BSA (51.3 ± 6.7% in the Z-VAD-fmk treated as compared with 55.9 ± 7.7% in controls). E, caspase-3 activity measured with the FRET-based sensor Casper 3-GR, expressed as ratio GFP/FRET of randomly selected cells under control conditions (BSA, white column, n = 3, 81 cells) and after cell treatment with 0.5 mm PA for 16–18 h (n = 3, 102 cells). F, histograms of cell cycle analysis by flow cytometry for DNA hypoploidy. Cells were treated with BSA or 0.5 mm PA and were analyzed after 18 h of incubation. G, statistical analysis of the histograms shown in panel F. H, statistical data representing nmol of ATP/mg of protein in the cells exposed to 0.5 mm PA (black circles, dotted line, n = 6 for each time point) or BSA alone (white circles, continuous line, n = 6 for each time point). *, p < 0.05 versus BSA.
Mentions: First we tested the susceptibility of the endothelial cell line, EA.hy926, to PA-induced cell death. For this purpose cells were treated with a complex of PA and BSA and cell viability was measured with the MTT assay at different times of incubation (Fig. 1A). After 12 h of incubation with PA, cell viability was found to be already significantly reduced and further decreased with time. The cell viability declined more than 80% after 28 h of incubation with PA (Fig. 1A), pointing to a strong cytotoxic effect of this saturated fatty acid on endothelial cells. The PA-induced cell death was further examined using long-term phase-contrast imaging, which showed an impaired cell proliferation and an accumulation of detached, dying cells upon incubation with PA (supplemental Fig. S1 and Movie S1). In contrast, cell treatment with OA did not affect the viability of the cells (Fig. 1A). These findings are in line with earlier studies using other cell types (26) and approved EA.hy926 cells as a suitable model to explore the molecular mechanisms of PA-mediated lipotoxicity.

Bottom Line: Here, we show that PA triggers autophagy, which did not counteract but in contrast promoted endothelial cell death.Moreover, the initiation of autophagy and cell death by PA was reduced in endothelial cells loaded with the Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-(acetoxymethyl) ester (BAPTA-AM), indicating that Ca(2+) triggers the fatal signaling of PA.In summary, we introduce an unexpected mechanism of lipotoxicity in endothelial cells and provide several novel strategies to counteract the lipotoxic signaling of PA.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, 8010 Graz, Austria.

ABSTRACT
Accumulation of palmitic acid (PA) in cells from nonadipose tissues is known to induce lipotoxicity resulting in cellular dysfunction and death. The exact molecular pathways of PA-induced cell death are still mysterious. Here, we show that PA triggers autophagy, which did not counteract but in contrast promoted endothelial cell death. The PA-induced cell death was predominantly necrotic as indicated by annexin V and propidium iodide (PI) staining, absence of caspase activity, low levels of DNA hypoploidy, and an early ATP depletion. In addition PA induced a strong elevation of mRNA levels of ubiquitin carboxyl-terminal hydrolase (CYLD), a known mediator of necroptosis. Moreover, siRNA-mediated knockdown of CYLD significantly antagonized PA-induced necrosis of endothelial cells. In contrast, inhibition and knockdown of receptor interacting protein kinase 1 (RIPK1) had no effect on PA-induced necrosis, indicating the induction of a CYLD-dependent but RIPK1-independent cell death pathway. PA was recognized as a strong and early inducer of autophagy. The inhibition of autophagy by both pharmacological inhibitors and genetic knockdown of the autophagy-specific genes, vacuolar protein sorting 34 (VPS34), and autophagy-related protein 7 (ATG7), could rescue the PA-induced death of endothelial cells. Moreover, the initiation of autophagy and cell death by PA was reduced in endothelial cells loaded with the Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-(acetoxymethyl) ester (BAPTA-AM), indicating that Ca(2+) triggers the fatal signaling of PA. In summary, we introduce an unexpected mechanism of lipotoxicity in endothelial cells and provide several novel strategies to counteract the lipotoxic signaling of PA.

Show MeSH
Related in: MedlinePlus