Journal: Cell Communication and Signaling : CCS

Article Title: ER stress inhibition enhances formation of triacylglcerols and protects endothelial cells from lipotoxicity

doi: 10.1186/s12964-024-01682-y

Figure Lengend Snippet: Saturated fatty acids induce impairment of autophagy and induction of UPR in endothelial cells. ( a ) BODIPY 493/503 staining of lipid droplets. HUVEC were seeded on 8-well IBIDI chamber and treated for 16 h with 150 µM of FFAs coupled to 5% BSA or BSA alone as a control. The cells were fixed, stained with BODIPY 493/503, anti-VE-Cadherin antibody and DAPI. The samples were imaged using Zeiss AxioObserver. Scale bar represents 15 μm. (b) Quantification of BODIPY staining from a). n = 3. (c) Immunofluorescence analysis of CHOP expression in endothelial cells. HUVEC were seeded on glass coverslips and treated for 16 h with 150 µM palmitate (PA) or oleate (OA) in combination with 5 µM A922500 or 10 µM PF-06424439. Treated cells were fixed and stained with anti CHOP antibody and DAPI. Scale bar represents 25 μm. (d) Immunoblot analysis of HUVEC treated as in c). Cells were lysed in 2x sample buffer and analysed for expression of LC3B, p62, pp38 and CHOP. Vinculin was used as loading control. (e) Quantification of immunoblots from d). All samples were normalized to control condition. n = 3. GraphPad Prism software and one-way ANOVA with Tukey’s multiple comparison test were used for statistical analysis. Error bars represent standard deviation (SD)

Article Snippet: Primary human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (#CC-2519) and cultured in Endothelial Cell medium (ECM) from ScienCell (#1001) supplemented with 5% FCS (Fetal Calf Serum), Endothelial Cell Growth Supplement and penicilin/streptomycin antibiotics.

Techniques: Staining, Control, Immunofluorescence, Expressing, Western Blot, Software, Comparison, Standard Deviation

Journal: Cell Communication and Signaling : CCS

Article Title: ER stress inhibition enhances formation of triacylglcerols and protects endothelial cells from lipotoxicity

doi: 10.1186/s12964-024-01682-y

Figure Lengend Snippet: ER stress inhibition restores palmitate-dependent impairment of autophagy and induction of UPR in macro- and microvascular endothelial cells. ( a ) Immunofluorescence of the ER morphology in HUVEC treated with 150 μm of oleate (OA) or palmitate (PA) in combination with DMSO or 2.5 mM 4-PBA. Prior to treatment the cells were transfected with the ER-scarlet probe. Lipid droplets were stained using BODIPY 493/503. The samples were imaged using Zeiss AxioObserver. Scale bar represents 15 μm. (b) Immunoblot analysis of HUVEC treated for 16 h with 150 μm PA in combination with DMSO or 2.5 mM 4-PBA. Cells were lysed in 2x sample buffer and analysed for expression of LC3B, p62, CHOP and Grp78. GAPDH immunblot was used as a loading control. (c) Quantification of immunoblot results from (b). 20 images per condition were analyzed in total in 3 independent experiments. (d) Immunoflourescence analysis of CHOP and p62 expression in HUVEC. The cells were treated as in ( b ), fixed and stained with anti-CHOP and anti-p62 antibodies and DAPI. Scale bar represents 50 μm. (e) Quantification of immunofluorescence analysis depicted in ( d ). For CHOP quantification percentage of CHOP positive cells is displayed and for p62 mean fluorescence intensity (MFI) normalized to control sample. 5 images per condition were analyzed in each of 3 independent experiments. (f) Immunofluorescence analysis of CHOP and p62 expression in HMVEC. Sample preparation and imaging was performed as in ( d ). (g) Quantification of CHOP and p62 staining in HMVEC depicted in ( f ) was performed as described for HUVEC in ( e ). ( i ) FITC labelled dextran leakage assay was performed in HUVEC. Cells were seeded on Transwell inserts and treated for 16 h with BSA + DMSO as a control, 150 µM PA + DMSO or 150 µM PA + 2.5 mM 4-PBA, 150 µM OA or 10 ng/ml TNFa as positive control. The leakage of FITC-labelled 75 kDa Dextran into lower compartment was measured by reading of mean fluorescence intensity at 488 nm using the microplate reader. 3 technical replicates were measured for each condition in 3 independent experiments. GraphPad Prism software and one-way ANOVA with Tukey’s multiple comparison test were used for statistical analysis. Error bars represent standard deviation (SD)

Article Snippet: Primary human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (#CC-2519) and cultured in Endothelial Cell medium (ECM) from ScienCell (#1001) supplemented with 5% FCS (Fetal Calf Serum), Endothelial Cell Growth Supplement and penicilin/streptomycin antibiotics.

Techniques: Inhibition, Immunofluorescence, Transfection, Staining, Western Blot, Expressing, Control, Fluorescence, Sample Prep, Imaging, Positive Control, Software, Comparison, Standard Deviation

Journal: Cell Communication and Signaling : CCS

Article Title: ER stress inhibition enhances formation of triacylglcerols and protects endothelial cells from lipotoxicity

doi: 10.1186/s12964-024-01682-y

Figure Lengend Snippet: 4-PBA co-treatment re-directs palmitate metabolism towards TG production and away from cholesterol synthesis. ( a ) Immunofluorescence analysis of PLIN2 expression in HUVEC treated with 150 µM palmitate (PA) alone or in combination with 2.5 mM 4-PBA for 16 h. As positive control the cells were treated with 150 µM oleate (OA) alone or in combination with 5 µM A922500. The samples were imaged using Zeiss AxioObserver. Scale bar represents 15 μm. (b) Quantification of PLIN2 staining from ( a ). Mean fluorescence intensity of PLIN2 was measured in 81–105 cells per condition in three independent experiments. Mean fluorescent intensity per cell normalized to control condition is shown in the graph. (c) Immunofluorescence staining of CHOP and PLIN2 in HUVEC treated with PA as in ( a ). CHOP is depicted in red, PLIN2 in magenta and actin staining in green. Scale bar represents 15 μm. Quantification of PLIN2 staining in CHOP positive ( n = 67) and CHOP negative ( n = 55) cells. Yellow arrows point towards representative CHOP + and CHOP – cells. (d) Quantification of total triglycerides (TG) in extract of HUVEC treated as in ( a ). Data obtained from three independent experiments are shown. (e) Representative images of free cholesterol staining in HUVEC treated as in ( a ). After the treatment, the cells were fixed and stained with 25 ug/ml of Fillipin III for 30 min and immediately imaged using Zeiss AxioObserver. Scale bar represents 50 μm. (f) Quantification of the Fillipin III staining from ( e ). Normalized mean fluorescence intensity of Fillipin III per cell is depicted. 5 images per condition were analyzed in three independent experiments. (g) Measurement of free cholesterol content in HUVEC treated as in ( a ). Free cholesterol was quantified using Cholesterol/Cholesteryl Ester Assay Kit in three independent experiments. GraphPad Prism software and one-way ANOVA with Tukey’s multiple comparison test or Student’s t-test were used for statistical analysis. Error bars represent standard deviation (SD)

Article Snippet: Primary human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (#CC-2519) and cultured in Endothelial Cell medium (ECM) from ScienCell (#1001) supplemented with 5% FCS (Fetal Calf Serum), Endothelial Cell Growth Supplement and penicilin/streptomycin antibiotics.

Techniques: Immunofluorescence, Expressing, Positive Control, Staining, Fluorescence, Control, Software, Comparison, Standard Deviation

Journal: Cell Communication and Signaling : CCS

Article Title: ER stress inhibition enhances formation of triacylglcerols and protects endothelial cells from lipotoxicity

doi: 10.1186/s12964-024-01682-y

Figure Lengend Snippet: Inhibition of ceramide synthesis reverts effects of palmitate in micro- and macrovascular endothelial cells. ( a ) Immunoflourescence analysis of CHOP and p62 expression in HUVEC. The cells were pre-treated with 500 nM myriocin for 8 h and then for additional 16 h with 150 µM palmitate (PA). Afterwards the cells were fixed and stained with anti-CHOP and anti-p62 antibodies and DAPI. (b) Quantification of CHOP and p62 immunofluorescence analysis in HUVEC. Percentage of CHOP positive HUVEC is depicted on the left and normalized mean fluorescence intensity (MFI) of p62 signal is shown on the right side. 5 images were analyzed per condition in three independent experiments. (c) Immunoflourescence analysis of CHOP and p62 expression in MVEC. The samples were prepared as in ( a ). (d) Quantification of CHOP and p62 immunofluorescence analysis in MVEC. Percentage of CHOP positive HUVEC is depicted on the left and normalized mean fluorescence intensity (MFI) of p62 signal per cell is shown on the right side. 5 images were analyzed per condition in three independent experiments. (e) Representative images of free cholesterol staining in HUVEC treated as in ( a ). After the treatment, the cells were stained with 25 ug/ml of Fillipin III for 30 min and immediately imaged using Zeiss AxioObserver. On the right side quantification of the Fillipin III staining is shown. 5 images were analyzed per condition in three independent experiments. (f) Representative images of free cholesterol staining in MVEC were obtained and analysed as in ( e ). Scale bars in all images represent 50 μm. GraphPad Prism software and one-way ANOVA with Tukey’s multiple comparison test or Student’s t-test were used for statistical analysis. Error bars represent standard deviation (SD)

Article Snippet: Primary human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (#CC-2519) and cultured in Endothelial Cell medium (ECM) from ScienCell (#1001) supplemented with 5% FCS (Fetal Calf Serum), Endothelial Cell Growth Supplement and penicilin/streptomycin antibiotics.

Techniques: Inhibition, Expressing, Staining, Immunofluorescence, Fluorescence, Software, Comparison, Standard Deviation

Journal: Cell Communication and Signaling : CCS

Article Title: ER stress inhibition enhances formation of triacylglcerols and protects endothelial cells from lipotoxicity

doi: 10.1186/s12964-024-01682-y

Figure Lengend Snippet: Palmitate induces synthesis of phosphatidic acid and enhances saturation of phospholipids in endothelial cells. ( a ) Principal component analysis (PCA) score plot of phospholipids measured in HUVEC treated with 150 μm palmitate (PA) alone or in combination with 2.5 mM 4-PBA or 500 nM myriocin. (b) PCA score plot of neutral lipid profiles obtained from HUVEC treated as in (a). (c) Acyl chain saturation in annotated phospholipids. Phospholipids were grouped according to the number of double bonds in their acyl chains. Within each group, the sums of normalized peak areas for each treatment condition are shown. (d) Acyl chain length in annotated phospholipids. Phospholipids were grouped according to the number of C atoms in their acyl chains. Within each group, the sums of normalized peak areas for each treatment condition are shown. (e) Normalized peak areas of total phosphatidic acid (PA) lipid class and most common annotated PA species (under). (f) Normalized peak areas of total phosphatydil inositol (PI) lipid class and most common annotated PI species (under). n = 3, GraphPad Prism software and one-way ANOVA with Tukey’s multiple comparison test were used for statistical analysis. Error bars represent standard deviation (SD)

Article Snippet: Primary human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (#CC-2519) and cultured in Endothelial Cell medium (ECM) from ScienCell (#1001) supplemented with 5% FCS (Fetal Calf Serum), Endothelial Cell Growth Supplement and penicilin/streptomycin antibiotics.

Techniques: Software, Comparison, Standard Deviation

Journal: Cell Communication and Signaling : CCS

Article Title: ER stress inhibition enhances formation of triacylglcerols and protects endothelial cells from lipotoxicity

doi: 10.1186/s12964-024-01682-y

Figure Lengend Snippet: ER stress inhibition switches palmitate metabolism in endothelial cells towards formation of TGs with saturated acyl chains. ( a ) Normalized peak areas of triglyceride (TG) fraction detected by mass spectrometry in HUVEC treated for 16 h with 150 μm palmitate (PA) alone or in combination with 2.5 mM 4-PBA or 500 nM myriocin. (b) Normalized peak areas of fragmented TG fraction detected in HUVEC treated as in ( (a) ). (c) Analysis of the acyl chain saturation in TG and fragmented TG fraction. TG and fragmented TG species were grouped according to the number of double bonds in their acyl chains. Within each group, the sums of normalized peak areas for each treatment condition are shown. (d) Normalized peak areas of fragmented TG fraction detected in HUVEC treated as in ( a ). n = 3, GraphPad Prism software and one-way ANOVA with Tukey’s multiple comparison test were used for statistical analysis. Error bars represent standard deviation (SD)

Article Snippet: Primary human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (#CC-2519) and cultured in Endothelial Cell medium (ECM) from ScienCell (#1001) supplemented with 5% FCS (Fetal Calf Serum), Endothelial Cell Growth Supplement and penicilin/streptomycin antibiotics.

Techniques: Inhibition, Mass Spectrometry, Software, Comparison, Standard Deviation

Journal: Cell Communication and Signaling : CCS

Article Title: ER stress inhibition enhances formation of triacylglcerols and protects endothelial cells from lipotoxicity

doi: 10.1186/s12964-024-01682-y

Figure Lengend Snippet: Induction of ceramide synthesis by palmitate in endothelial cells is blocked by 4-PBA. a ) Scheme of the sphingolipid synthesis pathway. Serine palmitoyltransferase (SPT), 3-ketodihydrosphingosine reductase (KSR), ceramide synthase (Cers), sphingolipid delta-4 desaturase (DEGS), sphingomyelin synthase (SMS) and glucosylceramide synthase (GCS). ( b ) Normalized peak areas of total ceramides detected by mass spectrometry in HUVEC treated for 16 h with 150 μm palmitate (PA) alone or in combination with 2.5 mM 4-PBA or 500 nM myriocin. ( c ) Normalized peak areas of most abundant ceramide species detected by lipidomic analysis. ( d ) Normalized peak areas of sphingomyelin lipid class detected by lipidomic analysis. ( e ) Normalized peak areas of glucosyl- and galactosylceramides detected in polar (left) or neutral (right side) lipid fraction. n = 3. GraphPad Prism software and one-way ANOVA with Tukey’s multiple comparison test were used for statistical analysis. Error bars represent standard deviation (SD)

Article Snippet: Primary human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (#CC-2519) and cultured in Endothelial Cell medium (ECM) from ScienCell (#1001) supplemented with 5% FCS (Fetal Calf Serum), Endothelial Cell Growth Supplement and penicilin/streptomycin antibiotics.

Techniques: Mass Spectrometry, Software, Comparison, Standard Deviation